Inhibitor compounds and cancer treatment methods

ABSTRACT

A synergistically effective combination of an anti-cancer agent and a therapeutic compound, such as an mTOR-Rictor complex inhibitor, a Serine 473 phosphorylation inhibitor, an AKT2 inhibitor, or a combination thereof, for use in the treatment of cancer, and methods and uses thereof. Also included are methods and uses of a thiosemicarbazone for treating a cancer in a mammal in need thereof characterized by over-expression of RAS, by an EGFR mutation, and/or by over-expression of AKT2.

FIELD OF THE INVENTION

The present invention relates generally to inhibitor compounds,compositions and cancer treatment methods.

BACKGROUND OF THE INVENTION

Cancer, irrespective of its pathogenesis, is characterized byuncontrolled growth and survival of cells. Common to most forms ofcancer is an error in the cellular mechanism responsible for balancingcell survival and cell death.

According to the American Cancer Society, lung cancer is the leadingcause of cancer death for both men and women. Small cell lung cancer(SCLC) accounts for approximately 20% of all lung cancers. The 5-yearsurvival rate for small cell lung cancer is about 15%.

Certain thiosemicarbazones, such as those disclosed in British PatentNo. 1,026,401, International Patent Application No. WO2004/066725,Japanese Patent No. 56-95161 and U.S. Pat. No. 4,927,843, have been usedto treat, for example, a variety of viruses. Other thiosemicarbazones,however, may be used to treat cancer. French Patent No. 2,879,194 isdirected to certain thiosemicarbazones that may be used in the treatmentor prevention of cancer, in dermatological treatment, in the treatmentof cardiovascular and immune diseases, lipid-metabolism related diseasesand modulate PPAR's. International Patent Application No. WO 2006/009765is directed to specific thiosemicarbazones that may be used inanti-cancer therapy that mitigates the development of drug resistance.U.S. Pat. No. 4,593,027 is directed to hydrazone derivatives that may beused as a chemotherapeutic.

There is a need, however, for new therapeutic drug treatments to treatcancers more efficiently, and lung cancer in particular. Currenttreatment regimes for small cell lung cancer involve surgery, radiationand chemotherapy. While timely surgery can be curative, new therapiesare necessary when timely surgery is not an option.

SUMMARY OF THE INVENTION

In an aspect, there is provided a therapeutically effective compositionfor use in the treatment of cancer comprising an anti-cancer agent and atherapeutically effective amount of a compound comprising a compound ofFormula I:

and/or a pharmaceutically-acceptable salt, hydrate, solvate, tautomer,optical isomer, or combination thereof;wherein:R₁ and R₂ together form a substituted or unsubstituted polycyclic ringcomprising at least two ring systems, said at least two ring systemscomprising a first ring system bonded to C1 and a second ring systemfused to the first ring system, wherein:the first ring system is a substituted or unsubstituted aromatic group,the second ring system is a substituted or unsubstituted aromatic group,a substituted or unsubstituted heteroaromatic group, a substituted orunsubstituted carbocyclic group, or a substituted or unsubstitutedheterocyclic group; orthe first ring system is a substituted or unsubstituted heteroaromaticgroup, the second ring system is a substituted or unsubstituted aromaticgroup, a substituted or unsubstituted heteroaromatic group, asubstituted or unsubstituted carbocyclic group, or a substituted orunsubstituted heterocyclic group; orthe first ring system is a substituted or unsubstituted saturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted unsaturatedcarbocyclic group, a substituted or unsubstituted heterocyclic group, ora substituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted unsaturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted carbocyclicgroup, a substituted or unsubstituted heterocyclic group, or asubstituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted heterocyclicgroup, the second ring system is a substituted or unsubstitutedheteroaromatic group, a substituted or unsubstituted carbocyclic group,or a substituted or unsubstituted heterocyclic group; andR₃ to R₁₁ are each independently selected from H, a substituted orunsubstituted hydrocarbon group, a substituted or unsubstitutedheterogeneous group, a substituted or unsubstituted carbocyclic group, asubstituted or unsubstituted heterocyclic group, substituted orunsubstituted aromatic, or a substituted or unsubstitutedheteroaromatic;R₁₂ is selected from H or a hydrocarbyl group;Y is selected from a heteroatom or a carbon atom;A is selected from a substituted or unsubstituted hydrocarbon group, asubstituted or unsubstituted heterogeneous group, a substituted orunsubstituted carbocyclic group, a substituted or unsubstitutedheterocyclic group, substituted or unsubstituted aromatic, or asubstituted or unsubstituted heteroaromatic; andn is an integer;wherein the composition produces a synergistic therapeutic effect ascompared to sole administration of either the anti-cancer agent or thecompound.

With respect to the above aspect, in another aspect, the compound is anmTOR-Rictor complex inhibitor, a Serine 473 phosphorylation inhibitor,an AKT2 inhibitor, or a combination thereof. In a further aspect, thecompound is an mTOR-Rictor complex inhibitor, a Serine 473phosphorylation inhibitor, or a combination thereof. In another aspect,the compound is both an mTOR-Rictor complex inhibitor and a Serine 473phosphorylation inhibitor. In another aspect, the compound is anmTOR-Rictor complex inhibitor. In another aspect, the anti-cancer agentis an mTOR-Raptor complex inhibitor. In another aspect, the anticanceragent is a cytotoxic agent. In another aspect, the synergistic effect isreduction or prevention of resistance to the cytotoxic agent. In anotheraspect, the anti-cancer agent is selected from the group consisting ofcisplatin, rapamycin, tecrolimus, temsirolimus, paclitaxel, erlotinib,cetuximab and doxorubicin. In another aspect, the cancer is treatable byinhibition of mTOR. In another aspect, the cancer is selected from thegroup consisting of lung cancer, cervical cancer, ovarian cancer, cancerof CNS, skin cancer, prostate cancer, sarcoma, breast cancer, leukemia,colorectal cancer, colon cancer, head cancer, neck cancer, endometrialcancer, and kidney cancer. In a further aspect, the cancer is selectedfrom the group consisting of small cell lung cancer, breast cancer,acute leukemia, chronic leukemia, colorectal cancer, colon cancer, braincancer, carcinoma, ovarian cancer, endometrial cancer, carcinoid tumorsmetastatic colorectal cancer, islet cell carcinoma, metastatic renalcell carcinoma adenocarcinomas, glioblastoma multiforme, bronchoalveolarlung cancers, non-Hodgkin's lymphoma, neuroendocrine tumors, andneuroblastoma. In another aspect, the cancer is ovarian, colon,colorectal or endometrial cancer. In another aspect, the amount of theanti-cancer agent is selected to lower overall toxicity as compared toadministration of the anti-cancer agent alone in an amount sufficient toachieve substantially the same treatment effect on cancerous cells. Inanother aspect, the dose of at least one of the anti-cancer agent or thecompound is selected to increase the overall treatment effect oncancerous cells as compared to administration of the anti-cancer agentalone in an amount producing substantially the same toxicity. In afurther aspect, the amount of the compound is less than an amount of theanti-cancer agent. In another aspect, the amount of the compound is atmost about 80%, about 50%, about 40%, about 30%, about 20%, about 10%,about 5%, about 3%, about 2%, about 1%, about 0.75%, about 0.5%, about0.25%, or about 0.1% of the amount of the anti-cancer agent. In anotheraspect, the first ring system is a substituted or unsubstitutedheterocyclic group, the second ring system is a substituted orunsubstituted heteroaromatic group, a substituted or unsubstitutedcarbocyclic group, or a substituted or unsubstituted heterocyclic group.In another aspect, the substituted or unsubstituted polycyclic ringfurther comprises a third ring system fused to the first ring system. Inanother aspect, the third ring system is a substituted or unsubstitutedaromatic group, a substituted or unsubstituted heteroaromatic group, asubstituted or unsubstituted carbocyclic group, or a substituted orunsubstituted heterocyclic group. In another aspect, n is 0. In anotheraspect, n is 1 and A is a substituted or unsubstituted heteroaromaticgroup. In another aspect, A is a pyridinyl group. In another aspect, Yis a nitrogen atom. In another aspect, R₇ is a substituted orunsubstituted alkyl group or a substituted or unsubstitutedheteroaromatic group and R₃ to R₆ and R₈ to R₁₂ are each independentlyselected from H or a substituted or unsubstituted hydrocarbon group. Inanother aspect, R₇ is the substituted or unsubstituted alkyl group or asubstituted or unsubstituted pyridyl group and R₃ to R₆ and R₈ to R₁₂are each H. In another aspect, the compound is selected from:

and/or a pharmaceutically-acceptable salt, hydrate, solvate orcombination thereof. In another aspect, the compound of Formula I is:

and/or a pharmaceutically-acceptable salt, hydrate, solvate orcombination thereof. In another aspect, the compound of Formula I is:

and/or a pharmaceutically-acceptable salt, hydrate, solvate orcombination thereof. In another aspect, the composition furthercomprising at least one pharmaceutically acceptable carrier and/ordiluent. In another aspect, the compound is apharmaceutically-acceptable salt of Formula I. In another aspect, thesalt is an oxalate or tartrate.

In yet another aspect, there is provided a pharmaceutically-acceptablesalt of a compound of Formula I:

and/or optical isomer thereof;wherein:R₁ and R₂ together form a substituted or unsubstituted polycyclic ringcomprising at least two ring systems, said at least two ring systemscomprising a first ring system bonded to C1 and a second ring systemfused to the first ring system, wherein:the first ring system is a substituted or unsubstituted aromatic group,the second ring system is a substituted or unsubstituted aromatic group,a substituted or unsubstituted heteroaromatic group, a substituted orunsubstituted carbocyclic group, or a substituted or unsubstitutedheterocyclic group; orthe first ring system is a substituted or unsubstituted heteroaromaticgroup, the second ring system is a substituted or unsubstituted aromaticgroup, a substituted or unsubstituted heteroaromatic group, asubstituted or unsubstituted carbocyclic group, or a substituted orunsubstituted heterocyclic group; orthe first ring system is a substituted or unsubstituted saturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted unsaturatedcarbocyclic group, a substituted or unsubstituted heterocyclic group, ora substituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted unsaturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted carbocyclicgroup, a substituted or unsubstituted heterocyclic group, or asubstituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted heterocyclicgroup, the second ring system is a substituted or unsubstitutedheteroaromatic group, a substituted or unsubstituted carbocyclic group,or a substituted or unsubstituted heterocyclic group; andR₃ to R₁₁ are each independently selected from H, a substituted orunsubstituted hydrocarbon group, a substituted or unsubstitutedheterogeneous group, a substituted or unsubstituted carbocyclic group, asubstituted or unsubstituted heterocyclic group, substituted orunsubstituted aromatic, or a substituted or unsubstitutedheteroaromatic;R₁₂ is selected from H or a hydrocarbyl group;Y is selected from a heteroatom or a carbon atom;A is selected from a substituted or unsubstituted hydrocarbon group, asubstituted or unsubstituted heterogeneous group, a substituted orunsubstituted carbocyclic group, a substituted or unsubstitutedheterocyclic group, substituted or unsubstituted aromatic, or asubstituted or unsubstituted heteroaromatic; andn is an integer.

In a further aspect, there is provided a pharmaceutically-acceptableoxalate or tartrate salt of a compound of Formula I:

and/or optical isomer thereof;wherein:R₁ and R₂ together form a substituted or unsubstituted polycyclic ringcomprising at least two ring systems, said at least two ring systemscomprising a first ring system bonded to C1 and a second ring systemfused to the first ring system, wherein:the first ring system is a substituted or unsubstituted aromatic group,the second ring system is a substituted or unsubstituted aromatic group,a substituted or unsubstituted heteroaromatic group, a substituted orunsubstituted carbocyclic group, or a substituted or unsubstitutedheterocyclic group; orthe first ring system is a substituted or unsubstituted heteroaromaticgroup, the second ring system is a substituted or unsubstituted aromaticgroup, a substituted or unsubstituted heteroaromatic group, asubstituted or unsubstituted carbocyclic group, or a substituted orunsubstituted heterocyclic group; orthe first ring system is a substituted or unsubstituted saturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted unsaturatedcarbocyclic group, a substituted or unsubstituted heterocyclic group, ora substituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted unsaturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted carbocyclicgroup, a substituted or unsubstituted heterocyclic group, or asubstituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted heterocyclicgroup, the second ring system is a substituted or unsubstitutedheteroaromatic group, a substituted or unsubstituted carbocyclic group,or a substituted or unsubstituted heterocyclic group; andR₃ to R₁₁ are each independently selected from H, a substituted orunsubstituted hydrocarbon group, a substituted or unsubstitutedheterogeneous group, a substituted or unsubstituted carbocyclic group, asubstituted or unsubstituted heterocyclic group, substituted orunsubstituted aromatic, or a substituted or unsubstitutedheteroaromatic;R₁₂ is selected from H or a hydrocarbyl group;Y is selected from a heteroatom or a carbon atom;A is selected from a substituted or unsubstituted hydrocarbon group, asubstituted or unsubstituted heterogeneous group, a substituted orunsubstituted carbocyclic group, a substituted or unsubstitutedheterocyclic group, substituted or unsubstituted aromatic, or asubstituted or unsubstituted heteroaromatic; andn is an integer.

With respect to the above aspects, in another aspect, the first ringsystem is a substituted or unsubstituted heterocyclic group, the secondring system is a substituted or unsubstituted heteroaromatic group, asubstituted or unsubstituted carbocyclic group, or a substituted orunsubstituted heterocyclic group. In another aspect, the substituted orunsubstituted polycyclic ring further comprises a third ring systemfused to the first ring system. In another aspect, the third ring systemis a substituted or unsubstituted aromatic group, a substituted orunsubstituted heteroaromatic group, a substituted or unsubstitutedcarbocyclic group, or a substituted or unsubstituted heterocyclic group.In another aspect, n is 0. In another aspect, n is 1 and A is asubstituted or unsubstituted heteroaromatic group. In another aspect, Ais a pyridinyl group. In another aspect, Y is a nitrogen atom. Inanother aspect, R₇ is a substituted or unsubstituted alkyl group or asubstituted or unsubstituted heteroaromatic group and R₃ to R₆ and R₈ toR₁₂ are each independently selected from H or a substituted orunsubstituted hydrocarbon group. In another aspect, R₇ is thesubstituted or unsubstituted alkyl group or a substituted orunsubstituted pyridyl group and R₃ to R₆ and R₈ to R₁₂ are each H. Inanother aspect, the compound is selected from the salt of:

In another aspect, the compound of Formula I is the salt of:

In another aspect, the compound of Formula I is the salt of:

In a further aspect, there is provided a method of treating a cancer ina mammal in need thereof comprising simultaneously or sequentiallyadministering an anti-cancer agent in combination with a therapeuticallyeffective amount of a compound that is an mTOR-Rictor complex inhibitor,a Serine 473 phosphorylation inhibitor, an AKT2 inhibitor, or acombination thereof, the combination providing a synergistic therapeuticeffect as compared to sole administration of either the anti-canceragent or the compound.

With respect to the above aspects, in another aspect, the cancer istreatable by inhibition of mTOR. In another aspect, the anti-canceragent is an mTOR-Raptor complex inhibitor. In another aspect, theanticancer agent is a cytotoxic agent. In another aspect, thesynergistic effect is reduction or prevention of resistance to thecytotoxic agent. In another aspect, the anti-cancer agent is selectedfrom the group consisting of cisplatin, rapamycin, tecrolimus,temsirolimus, paclitaxel, erlotinib, cetuximab and doxorubicin. Inanother aspect, the dose of the anti-cancer agent is selected to loweroverall toxicity as compared to administration of the anti-cancer agentalone in an amount sufficient to achieve substantially the sametreatment effect on cancerous cells. In another aspect, the dose of atleast one of the anti-cancer agent or the compound is selected toincrease the overall treatment effect on cancerous cells as compared toadministration of the anti-cancer agent alone in an amount producingsubstantially the same toxicity. In another aspect, the anti-canceragent is administered at a sub-therapeutic dose without substantiallyreducing the efficacy of the cancer treatment. In another aspect, thedose of the compound is less than the dose of the anti-cancer agent. Inanother aspect, the dose of the compound is at most about 80%, about50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 3%,about 2%, about 1%, about 0.75%, about 0.5%, about 0.25%, or about 0.1%of the dose of the anti-cancer agent. In another aspect, the compound isan mTOR-Rictor complex inhibitor. In another aspect, the cancer isselected from the group consisting of lung cancer, cervical cancer,ovarian cancer, cancer of CNS, skin cancer, prostate cancer, sarcoma,breast cancer, leukemia, colorectal cancer, colon cancer, head cancer,neck cancer, endometrial and kidney cancer. In another aspect, thecancer is selected from the group consisting of small cell lung cancer,breast cancer, acute leukemia, chronic leukemia, colorectal cancer,colon cancer, brain cancer, carcinoma, ovarian cancer, or endometrialcancer, carcinoid tumors, metatstatic colorectal cancer, islet cellcarcinoma, metastatic renal cell carcinoma, adenocarcinomas,glioblastoma multiforme, bronchoalveolar lung cancers, non-Hodgkin'slymphoma, neuroendocrine tumors, and neuroblastoma. In another aspect,the cancer is ovarian, colon, colorectal or endometrial cancer. Inanother aspect, the compound comprises a compound of Formula I:

and/or a pharmaceutically-acceptable salt, hydrate, solvate, tautomer,optical isomer, or combination thereof;wherein:R₁ and R₂ together form a substituted or unsubstituted polycyclic ringcomprising at least two ring systems, said at least two ring systemscomprising a first ring system bonded to C1 and a second ring systemfused to the first ring system, wherein:the first ring system is a substituted or unsubstituted aromatic group,the second ring system is a substituted or unsubstituted aromatic group,a substituted or unsubstituted heteroaromatic group, a substituted orunsubstituted carbocyclic group, or a substituted or unsubstitutedheterocyclic group; orthe first ring system is a substituted or unsubstituted heteroaromaticgroup, the second ring system is a substituted or unsubstituted aromaticgroup, a substituted or unsubstituted heteroaromatic group, asubstituted or unsubstituted carbocyclic group, or a substituted orunsubstituted heterocyclic group; orthe first ring system is a substituted or unsubstituted saturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted unsaturatedcarbocyclic group, a substituted or unsubstituted heterocyclic group, ora substituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted unsaturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted carbocyclicgroup, a substituted or unsubstituted heterocyclic group, or asubstituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted heterocyclicgroup, the second ring system is a substituted or unsubstitutedheteroaromatic group, a substituted or unsubstituted carbocyclic group,or a substituted or unsubstituted heterocyclic group; andR₃ to R₁₁ are each independently selected from H, a substituted orunsubstituted hydrocarbon group, a substituted or unsubstitutedheterogeneous group, a substituted or unsubstituted carbocyclic group, asubstituted or unsubstituted heterocyclic group, substituted orunsubstituted aromatic, or a substituted or unsubstitutedheteroaromatic;R₁₂ is selected from H or a hydrocarbyl group;Y is selected from a heteroatom or a carbon atom;A is selected from a substituted or unsubstituted hydrocarbon group, asubstituted or unsubstituted heterogeneous group, a substituted orunsubstituted carbocyclic group, a substituted or unsubstitutedheterocyclic group, substituted or unsubstituted aromatic, or asubstituted or unsubstituted heteroaromatic; andn is an integer.

In another aspect, there is provided a method of treating a cancer in amammal in need thereof comprising simultaneously or sequentiallyadministering an anti-cancer agent in combination with a therapeuticallyeffective amount of a compound comprising a compound of Formula I:

and/or a pharmaceutically-acceptable salt, hydrate, solvate, tautomer,optical isomer, or combination thereof;wherein:R₁ and R₂ together form a substituted or unsubstituted polycyclic ringcomprising at least two ring systems, said at least two ring systemscomprising a first ring system bonded to C1 and a second ring systemfused to the first ring system, wherein:the first ring system is a substituted or unsubstituted aromatic group,the second ring system is a substituted or unsubstituted aromatic group,a substituted or unsubstituted heteroaromatic group, a substituted orunsubstituted carbocyclic group, or a substituted or unsubstitutedheterocyclic group; orthe first ring system is a substituted or unsubstituted heteroaromaticgroup, the second ring system is a substituted or unsubstituted aromaticgroup, a substituted or unsubstituted heteroaromatic group, asubstituted or unsubstituted carbocyclic group, or a substituted orunsubstituted heterocyclic group; orthe first ring system is a substituted or unsubstituted saturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted unsaturatedcarbocyclic group, a substituted or unsubstituted heterocyclic group, ora substituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted unsaturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted carbocyclicgroup, a substituted or unsubstituted heterocyclic group, or asubstituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted heterocyclicgroup, the second ring system is a substituted or unsubstitutedheteroaromatic group, a substituted or unsubstituted carbocyclic group,or a substituted or unsubstituted heterocyclic group; andR₃ to R₁₁ are each independently selected from H, a substituted orunsubstituted hydrocarbon group, a substituted or unsubstitutedheterogeneous group, a substituted or unsubstituted carbocyclic group, asubstituted or unsubstituted heterocyclic group, substituted orunsubstituted aromatic, or a substituted or unsubstitutedheteroaromatic;R₁₂ is selected from H or a hydrocarbyl group;Y is selected from a heteroatom or a carbon atom;A is selected from a substituted or unsubstituted hydrocarbon group, asubstituted or unsubstituted heterogeneous group, a substituted orunsubstituted carbocyclic group, a substituted or unsubstitutedheterocyclic group, substituted or unsubstituted aromatic, or asubstituted or unsubstituted heteroaromatic; andn is an integer;the combination providing a synergistic therapeutic effect as comparedto sole administration of either the anti-cancer agent or the compound.

With respect to the above aspects, in another aspect, the cancer isselected from the group consisting of lung cancer, cervical cancer,ovarian cancer, cancer of CNS, skin cancer, prostate cancer, sarcoma,breast cancer, leukemia, colorectal cancer, colon cancer, head cancer,neck cancer, endometrial and kidney cancer. In another aspect, thecancer is selected from the group consisting of small cell lung cancer,breast cancer, acute leukemia, chronic leukemia, colorectal cancer,colon cancer, brain cancer, carcinoma, ovarian cancer, or endometrialcancer, carcinoid tumors, metastatic colorectal cancer, islet cellcarcinoma, metastatic renal cell carcinoma adenocarcinomas, glioblastomamultiforme, bronchoalveolar lung cancers, non-Hodgkin's lymphoma,neuroendocrine tumors, and neuroblastoma. In another aspect, the canceris ovarian, colon, colorectal or endometrial cancer.

In another aspect, there is provided a method of treating endometrialcancer in a mammal in need thereof comprising administering atherapeutically effective amount of a compound comprising a compound ofFormula I:

and/or a pharmaceutically-acceptable salt, hydrate, solvate, tautomer,optical isomer, or combination thereof;wherein:R₁ and R₂ together form a substituted or unsubstituted polycyclic ringcomprising at least two ring systems, said at least two ring systemscomprising a first ring system bonded to C1 and a second ring systemfused to the first ring system, wherein:the first ring system is a substituted or unsubstituted aromatic group,the second ring system is a substituted or unsubstituted aromatic group,a substituted or unsubstituted heteroaromatic group, a substituted orunsubstituted carbocyclic group, or a substituted or unsubstitutedheterocyclic group; orthe first ring system is a substituted or unsubstituted heteroaromaticgroup, the second ring system is a substituted or unsubstituted aromaticgroup, a substituted or unsubstituted heteroaromatic group, asubstituted or unsubstituted carbocyclic group, or a substituted orunsubstituted heterocyclic group; orthe first ring system is a substituted or unsubstituted saturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted unsaturatedcarbocyclic group, a substituted or unsubstituted heterocyclic group, ora substituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted unsaturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted carbocyclicgroup, a substituted or unsubstituted heterocyclic group, or asubstituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted heterocyclicgroup, the second ring system is a substituted or unsubstitutedheteroaromatic group, a substituted or unsubstituted carbocyclic group,or a substituted or unsubstituted heterocyclic group; andR₃ to R₁₁ are each independently selected from H, a substituted orunsubstituted hydrocarbon group, a substituted or unsubstitutedheterogeneous group, a substituted or unsubstituted carbocyclic group, asubstituted or unsubstituted heterocyclic group, substituted orunsubstituted aromatic, or a substituted or unsubstitutedheteroaromatic;R₁₂ is selected from H or a hydrocarbyl group;Y is selected from a heteroatom or a carbon atom;A is selected from a substituted or unsubstituted hydrocarbon group, asubstituted or unsubstituted heterogeneous group, a substituted orunsubstituted carbocyclic group, a substituted or unsubstitutedheterocyclic group, substituted or unsubstituted aromatic, or asubstituted or unsubstituted heteroaromatic; andn is an integer.

With respect to the above aspects, in another aspect, the cancer ischaracterized by a KRAS mutation. In another aspect, the cancer ischaracterized by an EGFR mutation.

In another aspect, there is provided a method of treating a cancer in amammal in need thereof characterized by over-expression of RAS, by anEGFR mutation, and/or by over-expression of AKT2, comprisingadministering a therapeutically effective amount of a compoundcomprising a compound of Formula I:

and/or a pharmaceutically-acceptable salt, hydrate, solvate, tautomer,optical isomer, or combination thereof;wherein:R₁ and R₂ together form a substituted or unsubstituted polycyclic ringcomprising at least two ring systems, said at least two ring systemscomprising a first ring system bonded to C1 and a second ring systemfused to the first ring system, wherein:the first ring system is a substituted or unsubstituted aromatic group,the second ring system is a substituted or unsubstituted aromatic group,a substituted or unsubstituted heteroaromatic group, a substituted orunsubstituted carbocyclic group, or a substituted or unsubstitutedheterocyclic group; orthe first ring system is a substituted or unsubstituted heteroaromaticgroup, the second ring system is a substituted or unsubstituted aromaticgroup, a substituted or unsubstituted heteroaromatic group, asubstituted or unsubstituted carbocyclic group, or a substituted orunsubstituted heterocyclic group; orthe first ring system is a substituted or unsubstituted saturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted unsaturatedcarbocyclic group, a substituted or unsubstituted heterocyclic group, ora substituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted unsaturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted carbocyclicgroup, a substituted or unsubstituted heterocyclic group, or asubstituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted heterocyclicgroup, the second ring system is a substituted or unsubstitutedheteroaromatic group, a substituted or unsubstituted carbocyclic group,or a substituted or unsubstituted heterocyclic group; andR₃ to R₁₁ are each independently selected from H, a substituted orunsubstituted hydrocarbon group, a substituted or unsubstitutedheterogeneous group, a substituted or unsubstituted carbocyclic group, asubstituted or unsubstituted heterocyclic group, substituted orunsubstituted aromatic, or a substituted or unsubstitutedheteroaromatic;R₁₂ is selected from H or a hydrocarbyl group;Y is selected from a heteroatom or a carbon atom;A is selected from a substituted or unsubstituted hydrocarbon group, asubstituted or unsubstituted heterogeneous group, a substituted orunsubstituted carbocyclic group, a substituted or unsubstitutedheterocyclic group, substituted or unsubstituted aromatic, or asubstituted or unsubstituted heteroaromatic; andn is an integer.

With respect to the above aspects, in another aspect, the cancer ischaracterized by a KRAS mutation. In another aspect, the cancer isselected from the group consisting of leukemia, colon cancer, colorectalcancer, pancreatic cancer, lung cancer, multiple myeloma, endometrialcancer, and ovarian cancer. In another aspect, the cancer is colorectalcancer. In another aspect, the cancer is characterized by an EGFRmutation. In another aspect, the cancer is selected from the groupconsisting of lung cancer, glioblastoma, colon cancer, gastric cancer,renal cancer, prostate cancer, breast cancer, and ovarian cancer. Inanother aspect, the cancer is non-small cell lung cancer. In anotheraspect, the cancer is characterized by over-expression of AKT2. Inanother aspect, the cancer is selected from the group consisting ofbreast cancer, ovarian cancer, colon cancer, pancreatic cancer, glioma,glioblastoma, lung cancer, and prostate cancer. In another aspect, thecancer is treatable by inhibition of mTOR. In another aspect, the methodfurther comprises simultaneously or sequentially administering ananti-cancer agent in combination with the compound. In another aspect,the combination produces a synergistic therapeutic effect as compared tosole administration of either the anti-cancer agent or the compound. Inanother aspect, the anti-cancer agent is an mTOR-Raptor complexinhibitor. In another aspect, the anticancer agent is a cytotoxic agent.In another aspect, the synergistic effect is reduction or prevention ofresistance to the cytotoxic agent. In another aspect, the anti-canceragent is selected from the group consisting of cisplatin, rapamycin,tecrolimus, temsirolimus, paclitaxel, erlotinib, cetuximab anddoxorubicin. In another aspect, the dose of the anti-cancer agent isselected to lower overall toxicity as compared to administration of theanti-cancer agent alone in an amount sufficient to achieve substantiallythe same treatment effect on cancerous cells. In another aspect, thedose of at least one of the anti-cancer agent or the compound isselected to increase the overall treatment effect on cancerous cells ascompared to administration of the anti-cancer agent alone in an amountproducing substantially the same toxicity. In another aspect, theanti-cancer agent is administered at a sub-therapeutic dose withoutsubstantially reducing the efficacy of the cancer treatment. In anotheraspect, the dose of the compound is less than the dose of theanti-cancer agent. In another aspect, the dose of the compound is atmost about 80%, about 50%, about 40%, about 30%, about 20%, about 10%,about 5%, about 3%, about 2%, about 1%, about 0.75%, about 0.5%, about0.25%, or about 0.1% of the dose of the anti-cancer agent. In anotheraspect, the first ring system is a substituted or unsubstitutedheterocyclic group, the second ring system is a substituted orunsubstituted heteroaromatic group, a substituted or unsubstitutedcarbocyclic group, or a substituted or unsubstituted heterocyclic group.In another aspect, the substituted or unsubstituted polycyclic ringfurther comprises a third ring system fused to the first ring system. Inanother aspect, the third ring system is a substituted or unsubstitutedaromatic group, a substituted or unsubstituted heteroaromatic group, asubstituted or unsubstituted carbocyclic group, or a substituted orunsubstituted heterocyclic group. In another aspect, n is 0. In anotheraspect, n is 1 and A is a substituted or unsubstituted heteroaromaticgroup. In another aspect, A is a pyridinyl group. In another aspect, Yis a nitrogen atom. In another aspect, R₇ is a substituted orunsubstituted alkyl group or a substituted or unsubstitutedheteroaromatic group and R₃ to R₆ and R₈ to R₁₂ are each independentlyselected from H or a substituted or unsubstituted hydrocarbon group. Inanother aspect, R₇ is the substituted or unsubstituted alkyl group or asubstituted or unsubstituted pyridyl group and R₃ to R₆ and R₈ to R₁₂are each H. In another aspect, the compound is selected from:

and/or a pharmaceutically-acceptable salt, hydrate, solvate orcombination thereof. In another aspect, the compound of Formula I is:

and/or a pharmaceutically-acceptable salt, hydrate, solvate orcombination thereof. In another aspect, the compound of Formula I is:

and/or a pharmaceutically-acceptable salt, hydrate, solvate orcombination thereof. In another aspect, the compound is apharmaceutically-acceptable salt of Formula I. In another aspect, thesalt is an oxalate or tartrate.

In another aspect, there is provided use of an anti-cancer agent incombination with a therapeutically effective amount of a compound thatis an mTOR-Rictor complex inhibitor, a Serine 473 phosphorylationinhibitor, an AKT2 inhibitor, or a combination thereof for treating acancer in a mammal in need thereof, wherein the combination provides asynergistic therapeutic effect as compared to sole administration ofeither the anti-cancer agent or the compound.

In another aspect, there is provided use of an anti-cancer agent incombination with a therapeutically effective amount of a compound thatis an mTOR-Rictor complex inhibitor, a Serine 473 phosphorylationinhibitor, an AKT2 inhibitor, or a combination thereof in themanufacture of a medicament for treating a cancer in a mammal in needthereof, wherein the combination provides a synergistic therapeuticeffect as compared to sole administration of either the anti-canceragent or the compound.

With respect to the above aspects, in another aspect, the cancer istreatable by inhibition of mTOR. In another aspect, the anti-canceragent is an mTOR-Raptor complex inhibitor. In another aspect, theanticancer agent is a cytotoxic agent. In another aspect, thesynergistic effect is reduction or prevention of resistance to thecytotoxic agent. In another aspect, the anti-cancer agent is selectedfrom the group consisting of cisplatin, rapamycin, tecrolimus,temsirolimus, paclitaxel, erlotinib, cetuximab and doxorubicin. Inanother aspect, the dose of the anti-cancer agent is selected to loweroverall toxicity as compared to administration of the anti-cancer agentalone in an amount sufficient to achieve substantially the sametreatment effect on cancerous cells. In another aspect, the dose of atleast one of the anti-cancer agent or the compound is selected toincrease the overall treatment effect on cancerous cells as compared toadministration of the anti-cancer agent alone in an amount producingsubstantially the same toxicity. In another aspect, the anti-canceragent is administered at a sub-therapeutic dose without substantiallyreducing the efficacy of the cancer treatment. In another aspect, thedose of the compound is less than the dose of the anti-cancer agent. Inanother aspect, the dose of the compound is at most about 80%, about50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 3%,about 2%, about 1%, about 0.75%, about 0.5%, about 0.25%, or about 0.1%of the dose of the anti-cancer agent. In another aspect, the compound isan mTOR-Rictor complex inhibitor. In another aspect, the cancer isselected from the group consisting of lung cancer, cervical cancer,ovarian cancer, cancer of CNS, skin cancer, prostate cancer, sarcoma,breast cancer, leukemia, colorectal cancer, colon cancer, head cancer,neck cancer, endometrial and kidney cancer. In another aspect, thecancer is selected from the group consisting of small cell lung cancer,breast cancer, acute leukemia, chronic leukemia, colorectal cancer,colon cancer, brain cancer, carcinoma, ovarian cancer, or endometrialcancer, carcinoid tumors, metastatic colorectal cancer, islet cellcarcinoma, metastatic renal cell carcinoma, adenocarcinomas,glioblastoma multiforme, bronchoalveolar lung cancers, non-Hodgkin'slymphoma, neuroendocrine tumors, and neuroblastoma. In another aspect,the cancer is ovarian, colon, colorectal or endometrial cancer. Inanother aspect, the compound comprises a compound of Formula I:

and/or a pharmaceutically-acceptable salt, hydrate, solvate, tautomer,optical isomer, or combination thereof;wherein:R₁ and R₂ together form a substituted or unsubstituted polycyclic ringcomprising at least two ring systems, said at least two ring systemscomprising a first ring system bonded to C1 and a second ring systemfused to the first ring system, wherein:the first ring system is a substituted or unsubstituted aromatic group,the second ring system is a substituted or unsubstituted aromatic group,a substituted or unsubstituted heteroaromatic group, a substituted orunsubstituted carbocyclic group, or a substituted or unsubstitutedheterocyclic group; orthe first ring system is a substituted or unsubstituted heteroaromaticgroup, the second ring system is a substituted or unsubstituted aromaticgroup, a substituted or unsubstituted heteroaromatic group, asubstituted or unsubstituted carbocyclic group, or a substituted orunsubstituted heterocyclic group; orthe first ring system is a substituted or unsubstituted saturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted unsaturatedcarbocyclic group, a substituted or unsubstituted heterocyclic group, ora substituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted unsaturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted carbocyclicgroup, a substituted or unsubstituted heterocyclic group, or asubstituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted heterocyclicgroup, the second ring system is a substituted or unsubstitutedheteroaromatic group, a substituted or unsubstituted carbocyclic group,or a substituted or unsubstituted heterocyclic group; andR₃ to R₁₁ are each independently selected from H, a substituted orunsubstituted hydrocarbon group, a substituted or unsubstitutedheterogeneous group, a substituted or unsubstituted carbocyclic group, asubstituted or unsubstituted heterocyclic group, substituted orunsubstituted aromatic, or a substituted or unsubstitutedheteroaromatic;R₁₂ is selected from H or a hydrocarbyl group;Y is selected from a heteroatom or a carbon atom;A is selected from a substituted or unsubstituted hydrocarbon group, asubstituted or unsubstituted heterogeneous group, a substituted orunsubstituted carbocyclic group, a substituted or unsubstitutedheterocyclic group, substituted or unsubstituted aromatic, or asubstituted or unsubstituted heteroaromatic; andn is an integer.

In another aspect, there is provided use of an anti-cancer agent incombination with a therapeutically effective amount of a compoundcomprising a compound of Formula I:

and/or a pharmaceutically-acceptable salt, hydrate, solvate, tautomer,optical isomer, or combination thereof;wherein:R₁ and R₂ together form a substituted or unsubstituted polycyclic ringcomprising at least two ring systems, said at least two ring systemscomprising a first ring system bonded to C1 and a second ring systemfused to the first ring system, wherein:the first ring system is a substituted or unsubstituted aromatic group,the second ring system is a substituted or unsubstituted aromatic group,a substituted or unsubstituted heteroaromatic group, a substituted orunsubstituted carbocyclic group, or a substituted or unsubstitutedheterocyclic group; orthe first ring system is a substituted or unsubstituted heteroaromaticgroup, the second ring system is a substituted or unsubstituted aromaticgroup, a substituted or unsubstituted heteroaromatic group, asubstituted or unsubstituted carbocyclic group, or a substituted orunsubstituted heterocyclic group; orthe first ring system is a substituted or unsubstituted saturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted unsaturatedcarbocyclic group, a substituted or unsubstituted heterocyclic group, ora substituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted unsaturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted carbocyclicgroup, a substituted or unsubstituted heterocyclic group, or asubstituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted heterocyclicgroup, the second ring system is a substituted or unsubstitutedheteroaromatic group, a substituted or unsubstituted carbocyclic group,or a substituted or unsubstituted heterocyclic group; andR₃ to R₁₁ are each independently selected from H, a substituted orunsubstituted hydrocarbon group, a substituted or unsubstitutedheterogeneous group, a substituted or unsubstituted carbocyclic group, asubstituted or unsubstituted heterocyclic group, substituted orunsubstituted aromatic, or a substituted or unsubstitutedheteroaromatic;R₁₂ is selected from H or a hydrocarbyl group;Y is selected from a heteroatom or a carbon atom;A is selected from a substituted or unsubstituted hydrocarbon group, asubstituted or unsubstituted heterogeneous group, a substituted orunsubstituted carbocyclic group, a substituted or unsubstitutedheterocyclic group, substituted or unsubstituted aromatic, or asubstituted or unsubstituted heteroaromatic; andn is an integer;for treating a cancer in a mammal in need thereof, wherein thecombination provides a synergistic therapeutic effect as compared tosole administration of either the anti-cancer agent or the compound.

In another aspect, there is provided use of an anti-cancer agent incombination with a therapeutically effective amount of a compoundcomprising a compound of Formula I:

and/or a pharmaceutically-acceptable salt, hydrate, solvate, tautomer,optical isomer, or combination thereof;wherein:R₁ and R₂ together form a substituted or unsubstituted polycyclic ringcomprising at least two ring systems, said at least two ring systemscomprising a first ring system bonded to C1 and a second ring systemfused to the first ring system, wherein:the first ring system is a substituted or unsubstituted aromatic group,the second ring system is a substituted or unsubstituted aromatic group,a substituted or unsubstituted heteroaromatic group, a substituted orunsubstituted carbocyclic group, or a substituted or unsubstitutedheterocyclic group; orthe first ring system is a substituted or unsubstituted heteroaromaticgroup, the second ring system is a substituted or unsubstituted aromaticgroup, a substituted or unsubstituted heteroaromatic group, asubstituted or unsubstituted carbocyclic group, or a substituted orunsubstituted heterocyclic group; orthe first ring system is a substituted or unsubstituted saturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted unsaturatedcarbocyclic group, a substituted or unsubstituted heterocyclic group, ora substituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted unsaturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted carbocyclicgroup, a substituted or unsubstituted heterocyclic group, or asubstituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted heterocyclicgroup, the second ring system is a substituted or unsubstitutedheteroaromatic group, a substituted or unsubstituted carbocyclic group,or a substituted or unsubstituted heterocyclic group; andR₃ to R₁₁ are each independently selected from H, a substituted orunsubstituted hydrocarbon group, a substituted or unsubstitutedheterogeneous group, a substituted or unsubstituted carbocyclic group, asubstituted or unsubstituted heterocyclic group, substituted orunsubstituted aromatic, or a substituted or unsubstitutedheteroaromatic;R₁₂ is selected from H or a hydrocarbyl group;Y is selected from a heteroatom or a carbon atom;A is selected from a substituted or unsubstituted hydrocarbon group, asubstituted or unsubstituted heterogeneous group, a substituted orunsubstituted carbocyclic group, a substituted or unsubstitutedheterocyclic group, substituted or unsubstituted aromatic, or asubstituted or unsubstituted heteroaromatic; andn is an integer;in the manufacture of a medicament for treating a cancer in a mammal inneed thereof, wherein the combination provides a synergistic therapeuticeffect as compared to sole administration of either the anti-canceragent or the compound.

With respect to the above aspects, in another aspect, the cancer isselected from the group consisting of lung cancer, cervical cancer,ovarian cancer, cancer of CNS, skin cancer, prostate cancer, sarcoma,breast cancer, leukemia, colorectal cancer, colon cancer, head cancer,neck cancer, endometrial and kidney cancer. In another aspect, thecancer is selected from the group consisting of small cell lung cancer,breast cancer, acute leukemia, chronic leukemia, colorectal cancer,colon cancer, brain cancer, carcinoma, ovarian cancer, or endometrialcancer, carcinoid tumors, metastatic colorectal cancer, islet cellcarcinoma, metastatic renal cell carcinoma, adenocarcinomas,glioblastoma multiforme, bronchoalveolar lung cancers, non-Hodgkin'slymphoma, neuroendocrine tumors, and neuroblastoma. In another aspect,the cancer is ovarian, colon, colorectal or endometrial cancer.

In another aspect, there is provided use of a therapeutically effectiveamount of a compound comprising a compound of Formula I:

and/or a pharmaceutically-acceptable salt, hydrate, solvate, tautomer,optical isomer, or combination thereof;wherein:R₁ and R₂ together form a substituted or unsubstituted polycyclic ringcomprising at least two ring systems, said at least two ring systemscomprising a first ring system bonded to C1 and a second ring systemfused to the first ring system, wherein:the first ring system is a substituted or unsubstituted aromatic group,the second ring system is a substituted or unsubstituted aromatic group,a substituted or unsubstituted heteroaromatic group, a substituted orunsubstituted carbocyclic group, or a substituted or unsubstitutedheterocyclic group; orthe first ring system is a substituted or unsubstituted heteroaromaticgroup, the second ring system is a substituted or unsubstituted aromaticgroup, a substituted or unsubstituted heteroaromatic group, asubstituted or unsubstituted carbocyclic group, or a substituted orunsubstituted heterocyclic group; orthe first ring system is a substituted or unsubstituted saturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted unsaturatedcarbocyclic group, a substituted or unsubstituted heterocyclic group, ora substituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted unsaturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted carbocyclicgroup, a substituted or unsubstituted heterocyclic group, or asubstituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted heterocyclicgroup, the second ring system is a substituted or unsubstitutedheteroaromatic group, a substituted or unsubstituted carbocyclic group,or a substituted or unsubstituted heterocyclic group; andR₃ to R₁₁ are each independently selected from H, a substituted orunsubstituted hydrocarbon group, a substituted or unsubstitutedheterogeneous group, a substituted or unsubstituted carbocyclic group, asubstituted or unsubstituted heterocyclic group, substituted orunsubstituted aromatic, or a substituted or unsubstitutedheteroaromatic;R₁₂ is selected from H or a hydrocarbyl group;Y is selected from a heteroatom or a carbon atom;A is selected from a substituted or unsubstituted hydrocarbon group, asubstituted or unsubstituted heterogeneous group, a substituted orunsubstituted carbocyclic group, a substituted or unsubstitutedheterocyclic group, substituted or unsubstituted aromatic, or asubstituted or unsubstituted heteroaromatic; andn is an integer,for treating endometrial cancer in a mammal in need thereof.

In another aspect, there is provided use of a therapeutically effectiveamount of a compound comprising a compound of Formula I:

and/or a pharmaceutically-acceptable salt, hydrate, solvate, tautomer,optical isomer, or combination thereof;wherein:R₁ and R₂ together form a substituted or unsubstituted polycyclic ringcomprising at least two ring systems, said at least two ring systemscomprising a first ring system bonded to C1 and a second ring systemfused to the first ring system, wherein:the first ring system is a substituted or unsubstituted aromatic group,the second ring system is a substituted or unsubstituted aromatic group,a substituted or unsubstituted heteroaromatic group, a substituted orunsubstituted carbocyclic group, or a substituted or unsubstitutedheterocyclic group; orthe first ring system is a substituted or unsubstituted heteroaromaticgroup, the second ring system is a substituted or unsubstituted aromaticgroup, a substituted or unsubstituted heteroaromatic group, asubstituted or unsubstituted carbocyclic group, or a substituted orunsubstituted heterocyclic group; orthe first ring system is a substituted or unsubstituted saturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted unsaturatedcarbocyclic group, a substituted or unsubstituted heterocyclic group, ora substituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted unsaturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted carbocyclicgroup, a substituted or unsubstituted heterocyclic group, or asubstituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted heterocyclicgroup, the second ring system is a substituted or unsubstitutedheteroaromatic group, a substituted or unsubstituted carbocyclic group,or a substituted or unsubstituted heterocyclic group; andR₃ to R₁₁ are each independently selected from H, a substituted orunsubstituted hydrocarbon group, a substituted or unsubstitutedheterogeneous group, a substituted or unsubstituted carbocyclic group, asubstituted or unsubstituted heterocyclic group, substituted orunsubstituted aromatic, or a substituted or unsubstitutedheteroaromatic;R₁₂ is selected from H or a hydrocarbyl group;Y is selected from a heteroatom or a carbon atom;A is selected from a substituted or unsubstituted hydrocarbon group, asubstituted or unsubstituted heterogeneous group, a substituted orunsubstituted carbocyclic group, a substituted or unsubstitutedheterocyclic group, substituted or unsubstituted aromatic, or asubstituted or unsubstituted heteroaromatic; andn is an integer,in the manufacture of a medicament for treating endometrial cancer in amammal in need thereof.

With respect to the above aspects, in another aspect, the cancer ischaracterized by a KRAS mutation. In another aspect, the cancer ischaracterized by an EGFR mutation.

In another aspect, there is provided use of a therapeutically effectiveamount of a compound comprising a compound of Formula I:

and/or a pharmaceutically-acceptable salt, hydrate, solvate, tautomer,optical isomer, or combination thereof;wherein:R₁ and R₂ together form a substituted or unsubstituted polycyclic ringcomprising at least two ring systems, said at least two ring systemscomprising a first ring system bonded to C1 and a second ring systemfused to the first ring system, wherein:the first ring system is a substituted or unsubstituted aromatic group,the second ring system is a substituted or unsubstituted aromatic group,a substituted or unsubstituted heteroaromatic group, a substituted orunsubstituted carbocyclic group, or a substituted or unsubstitutedheterocyclic group; orthe first ring system is a substituted or unsubstituted heteroaromaticgroup, the second ring system is a substituted or unsubstituted aromaticgroup, a substituted or unsubstituted heteroaromatic group, asubstituted or unsubstituted carbocyclic group, or a substituted orunsubstituted heterocyclic group; orthe first ring system is a substituted or unsubstituted saturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted unsaturatedcarbocyclic group, a substituted or unsubstituted heterocyclic group, ora substituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted unsaturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted carbocyclicgroup, a substituted or unsubstituted heterocyclic group, or asubstituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted heterocyclicgroup, the second ring system is a substituted or unsubstitutedheteroaromatic group, a substituted or unsubstituted carbocyclic group,or a substituted or unsubstituted heterocyclic group; andR₃ to R₁₁ are each independently selected from H, a substituted orunsubstituted hydrocarbon group, a substituted or unsubstitutedheterogeneous group, a substituted or unsubstituted carbocyclic group, asubstituted or unsubstituted heterocyclic group, substituted orunsubstituted aromatic, or a substituted or unsubstitutedheteroaromatic;R₁₂ is selected from H or a hydrocarbyl group;Y is selected from a heteroatom or a carbon atom;A is selected from a substituted or unsubstituted hydrocarbon group, asubstituted or unsubstituted heterogeneous group, a substituted orunsubstituted carbocyclic group, a substituted or unsubstitutedheterocyclic group, substituted or unsubstituted aromatic, or asubstituted or unsubstituted heteroaromatic; andn is an integer,for treating a cancer in a mammal in need thereof, the cancercharacterized by over-expression of RAS, by an EGFR mutation, and/or byover-expression of AKT2.

In another aspect, there is provided use of a therapeutically effectiveamount of a compound comprising a compound of Formula I:

and/or a pharmaceutically-acceptable salt, hydrate, solvate, tautomer,optical isomer, or combination thereof;wherein:R₁ and R₂ together form a substituted or unsubstituted polycyclic ringcomprising at least two ring systems, said at least two ring systemscomprising a first ring system bonded to C1 and a second ring systemfused to the first ring system, wherein:the first ring system is a substituted or unsubstituted aromatic group,the second ring system is a substituted or unsubstituted aromatic group,a substituted or unsubstituted heteroaromatic group, a substituted orunsubstituted carbocyclic group, or a substituted or unsubstitutedheterocyclic group; orthe first ring system is a substituted or unsubstituted heteroaromaticgroup, the second ring system is a substituted or unsubstituted aromaticgroup, a substituted or unsubstituted heteroaromatic group, asubstituted or unsubstituted carbocyclic group, or a substituted orunsubstituted heterocyclic group; orthe first ring system is a substituted or unsubstituted saturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted unsaturatedcarbocyclic group, a substituted or unsubstituted heterocyclic group, ora substituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted unsaturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted carbocyclicgroup, a substituted or unsubstituted heterocyclic group, or asubstituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; orthe first ring system is a substituted or unsubstituted heterocyclicgroup, the second ring system is a substituted or unsubstitutedheteroaromatic group, a substituted or unsubstituted carbocyclic group,or a substituted or unsubstituted heterocyclic group; andR₃ to R₁₁ are each independently selected from H, a substituted orunsubstituted hydrocarbon group, a substituted or unsubstitutedheterogeneous group, a substituted or unsubstituted carbocyclic group, asubstituted or unsubstituted heterocyclic group, substituted orunsubstituted aromatic, or a substituted or unsubstitutedheteroaromatic;R₁₂ is selected from H or a hydrocarbyl group;Y is selected from a heteroatom or a carbon atom;A is selected from a substituted or unsubstituted hydrocarbon group, asubstituted or unsubstituted heterogeneous group, a substituted orunsubstituted carbocyclic group, a substituted or unsubstitutedheterocyclic group, substituted or unsubstituted aromatic, or asubstituted or unsubstituted heteroaromatic; andn is an integer,in the manufacture of a medicament for treating a cancer in a mammal inneed thereof, the cancer characterized by over-expression of RAS, by anEGFR mutation, and/or by over-expression of AKT2.

With respect to the above aspects, in another aspect, the cancer ischaracterized by a KRAS mutation. In another aspect, the cancer isselected from the group consisting of leukemia, colon cancer, colorectalcancer, pancreatic cancer, lung cancer, multiple myeloma, endometrialcancer, and ovarian cancer. In another aspect, the cancer is colorectalcancer. In another aspect, the cancer is characterized by an EGFRmutation. In another aspect, the cancer is selected from the groupconsisting of lung cancer, glioblastoma, colon cancer, gastric cancer,renal cancer, prostate cancer, breast cancer, and ovarian cancer. Inanother aspect, the cancer is non-small cell lung cancer. In anotheraspect, the cancer is characterized by over-expression of AKT2. Inanother aspect, the cancer is selected from the group consisting ofbreast cancer, ovarian cancer, colon cancer, pancreatic cancer, glioma,glioblastoma, lung cancer, and prostate cancer. In another aspect, thecancer is treatable by inhibition of mTOR. In another aspect, the usefurther comprises an anti-cancer agent in combination with the compound.In another aspect, the combination produces a synergistic therapeuticeffect as compared to sole administration of either the anti-canceragent or the compound. In another aspect, the anti-cancer agent is anmTOR-Raptor complex inhibitor. In another aspect, the anticancer agentis a cytotoxic agent. In another aspect, the synergistic effect isreduction or prevention of resistance to the cytotoxic agent. In anotheraspect, the anti-cancer agent is selected from the group consisting ofcisplatin, rapamycin, tecrolimus, temsirolimus, paclitaxel, erlotinib,cetuximab and doxorubicin. In another aspect, the amount of theanti-cancer agent is selected to lower overall toxicity as compared toadministration of the anti-cancer agent alone in an amount sufficient toachieve substantially the same treatment effect on cancerous cells. Inanother aspect, the amount of at least one of the anti-cancer agent orthe compound is selected to increase the overall treatment effect oncancerous cells as compared to administration of the anti-cancer agentalone in an amount producing substantially the same toxicity. In anotheraspect, the anti-cancer agent is present at a sub-therapeutic amountwithout substantially reducing the efficacy of the cancer treatment. Inanother aspect, the amount of the compound is less than the amount ofthe anti-cancer agent. In another aspect, the amount of the compound isat most about 80%, about 50%, about 40%, about 30%, about 20%, about10%, about 5%, about 3%, about 2%, about 1%, about 0.75%, about 0.5%,about 0.25%, or about 0.1% of the amount of the anti-cancer agent. Inanother aspect, the first ring system is a substituted or unsubstitutedheterocyclic group, the second ring system is a substituted orunsubstituted heteroaromatic group, a substituted or unsubstitutedcarbocyclic group, or a substituted or unsubstituted heterocyclic group.In another aspect, the substituted or unsubstituted polycyclic ringfurther comprises a third ring system fused to the first ring system. Inanother aspect, the third ring system is a substituted or unsubstitutedaromatic group, a substituted or unsubstituted heteroaromatic group, asubstituted or unsubstituted carbocyclic group, or a substituted orunsubstituted heterocyclic group. In another aspect, n is 0. In anotheraspect, n is 1 and A is a substituted or unsubstituted heteroaromaticgroup. In another aspect, A is a pyridinyl group. In another aspect, Yis a nitrogen atom. In another aspect, R₇ is a substituted orunsubstituted alkyl group or a substituted or unsubstitutedheteroaromatic group and R₃ to R₆ and R₈ to R₁₂ are each independentlyselected from H or a substituted or unsubstituted hydrocarbon group. Inanother aspect, R₇ is the substituted or unsubstituted alkyl group or asubstituted or unsubstituted pyridyl group and R₃ to R₆ and R₈ to R₁₂are each H. In another aspect, the compound is selected from:

and/or a pharmaceutically-acceptable salt, hydrate, solvate orcombination thereof. In another aspect, the compound of Formula I is:

and/or a pharmaceutically-acceptable salt, hydrate, solvate orcombination thereof. In another aspect, the compound of Formula I is:

and/or a pharmaceutically-acceptable salt, hydrate, solvate orcombination thereof. In another aspect, the compound is apharmaceutically-acceptable salt of Formula I. In another aspect, thesalt is an oxalate or tartrate.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating embodiments of the invention are given by wayof illustration only, since various changes and modifications within thespirit and scope of the invention will become apparent to those skilledin the art from the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described, by way ofexample only, with reference to the attached Figures.

FIG. 1 shows the volume of SHP77 human SCLC tumour in nude mice treatedwith test compounds;

FIG. 2 shows number of SHP77 human SCLC tumours in nude mice treatedwith test compounds;

FIG. 3 shows the volume of N417 human SCLC tumour in nude mice treatedwith COTI-2 and control;

FIG. 4 shows lack of emerging resistance in DMS153 cells treated withCOTI-2 and COTI-219;

FIG. 5 shows lack of emerging resistance in SHP77 cells treated withCOTI-2 and COTI-219;

FIGS. 6A and 6B show volume of U87 human glioma tumours in nude micetreated with two different concentrations of COTI-2;

FIG. 7 shows Western blot analysis of cellular lysates of SHP77 cellsthat have been treated with COTI-2;

FIG. 8 shows inhibition of AKT kinase activity by COTI-2;

FIG. 9 shows a time course of AKT expression following incubation withCOTI-2;

FIG. 10 shows a percent change and direction of differentially expressedgenes;

FIG. 11A-11C show expression levels of AKT1 (FIG. 11A), AKT2 (FIG. 11B),and mTOR-Rictor (FIG. 11C) in DMS114 and SHP77 cell lines followingincubation for 6 h in the presence (150 or 300 nM) or absence of COTI-2;

FIG. 12 shows PIP3 levels in DMS114 and SHP77 cells following incubationwith 250 nM COTI-2 alone, 250 nM COTI-2+LY294002, or DMSO (solvent)alone;

FIG. 13 shows an effect of various concentrations of COTI-2 relative tocontrol (DMSO) on total mTOR, mTOR phospho-Ser2448, mTORphospho-Ser2481, and Akt phospho-Thr308 levels, β actin levels act aspositive controls for the experiment;

FIG. 14 shows that temsirolimus and rapamycin both inhibit proliferationof human U87 glioblastoma cells, as single agents;

FIG. 15 shows that COTI-219, in combination with temsirolimus, had agreater-than-additive inhibitory effect on proliferation of U87glioblastoma cells;

FIG. 16 shows that COTI-219, in combination with rapamycin, had agreater-than-additive inhibitory effect on proliferation of U87glioblastoma cells;

FIG. 17 shows that COTI-2, in combination with temsirolimus, had agreater-than-additive inhibitory effect on proliferation of U87glioblastoma cells;

FIG. 18 shows that COTI-2, in combination with rapamycin, had agreater-than-additive inhibitory effect on proliferation of U87glioblastoma cells;

FIG. 19 shows SHP 77 cells treated with Taxol™ or with Taxol™ plusCOTI-2;

FIG. 20 shows DMS114 cells treated with Taxol™ or with Taxol™ plusCOTI-2;

FIG. 21 shows DMS114 cells treated with cisplatin (CDDP) or with CDDPplus COTI-2;

FIG. 22 shows SHP 77 cells treated with cisplatin (CDDP) or with CDDPplus COTI-2;

FIG. 23 shows SHP 77 cells treated with carboplatin or with carboplatinplus COTI-2;

FIG. 24 shows DMS114 cells treated with carboplatin or with carboplatinplus COTI-2;

FIG. 25 shows SHP 77 cells treated with gemcitabine alone or withgemcitabine plus COTI-2;

FIG. 26 shows DMS114 cells treated with gemcitabine alone or withgemcitabine plus COTI-2;

FIG. 27 shows SHP 77 cells treated with vinorelbine alone or withvinorelbine plus COTI-2;

FIG. 28 shows DMS114 cells treated with vinorelbine alone or withvinorelbine plus COTI-2;

FIGS. 29A-C show greater-than-additive effects of COTI-2 in combinationwith Tarceva in HCT-15 (FIG. 29A), COLO-205 (FIG. 29B), and SW620 (FIG.29C) cells;

FIGS. 30A-C show greater-than-additive effects of COTI-2 in combinationwith Erbitux® in HCT-15 (FIG. 30A), COLO-205 (FIG. 30B), and SW620 cells(FIG. 30C);

FIGS. 31A-C show greater-than-additive effects of COTI-219 incombination with Tarceva® in HCT-15 (FIG. 31A), COLO-205 (FIG. 31B), andSW620 cells (FIG. 31C);

FIGS. 32A-C show greater-than-additive effects of COTI-219 incombination with Erbitux® in HCT-15 (FIG. 32A), COLO-205 (FIG. 32B), andSW620 cells (FIG. 32C);

FIG. 33 shows the correlation between AKT isoforms (1, 2, & 3)expression and COTI-2 IC50, lane numbers represent the ratios of AKTisoform expression relative to β-actin expression, and R valuesrepresent the correlation between normalized AKT levels and IC50 values;

FIG. 34 shows an effect of AKT/AKT2 siRNA on COTI-2 induced apoptosis inDMS114 SCLC cells, the asterisk (*) indicates statistical significance(p<0.05);

FIG. 35 shows the total AKT and AKT2 expression levels in DMS114 cellsincubated with 0-100 ng AKT2 siRNA;

FIG. 36 shows the percent viability of SHP77 cells transfected withscrambled, total AKT, or AKT2 siRNA and in the presence/absence ofvarious concentrations COTI-2, the asterisk (*) indicates statisticalsignificance (p<0.05);

FIG. 37 shows the percent viability of SHP77 cells in the presence ofscrambled, total AKT, or AKT2 siRNA, the asterisk (*) indicatesstatistical significance (p<0.05);

FIG. 38 shows the levels of phosphorylation of AKT at Ser473 site andGSK-3α/β at Ser21/9 site in the presence of 250 nM COTI-2 at 0, 2, 4, 8,and 16 h post-incubation, the levels of phospho-Ser473 of AKT wereconfirmed with antibodies from 2 providers, Cell Signaling and Abcam;

FIG. 39 shows the time-course of COTI-2 effect on AKT pSer473 in twoSCLC cell lines, DMS114 and SHP77;

FIG. 40 shows changes in AKT phospho-protein targets, total proteinlysates were obtained at different time points following incubation with300 nM of COTI-2, following SDS-PAGE separation of 50 μg of protein theblots were probed with the indicated antibodies;

FIGS. 41A and 41B show mTor pSer2481 and AKT pSer473 in DMS114, theblots shown in FIG. 37 were scanned and quantitated with ImageJsoftware, and phospho-protein expression was corrected to total mTORlevels;

FIGS. 42A and 42B show mTOR pSer2448/pSer2481 in SHP77 cells, the blotsshown in FIG. 40 were scanned and quantitated with ImageJ software, andphospho-protein expression was corrected to total mTOR levels;

FIGS. 43A and 43B show down-regulation of FOXO transcription factors,phospho-FOXO1 and phosphor-FOXO3a levels were measured by densitometryanalysis and corrected for total protein levels, as described in thelegend to FIG. 42;

FIG. 44 shows a western blot analysis of PTEN in HeLa cells transfectedwith PTEN targeted siRNA (+) and non-targeted siRNA (−);

FIGS. 45A and 45B shows reverse transcription PCR of HeLa (FIG. 45A) andMCF-7 cells (FIG. 45B) transfected with 100 nM targeted PTEN siRNA ornon-targeted siRNA, cells were evaluated at 0, 24, 48, and 144 hpost-transfection;

FIGS. 46A & 46B show the effect of knockdown of PTEN mRNA with PTENsiRNA on HeLa (FIG. 46A) and MCF-7 (FIG. 46B) cell sensitivity toCOTI-2;

FIG. 47 shows sensitivity of human tumor cell lines (H226 & HL-60) andprimary human peripheral blood mononuclear cells (PBMCs) to COTI2-M05,data are represented as the mean of 5 measurements±standard error andthe asterisk (*) indicates a significant difference from controls(p<0.05);

FIG. 48 shows representative western blot analysis of DMS114 and SHP77cells transfected with gene-specific siRNA or scrambled controloligonucleotide for 48 h;

FIGS. 49A-B show determination of apoptosis following target knock-downwith gene-specific or scrambled (sc) siRNA in the absence or presence of500 nM COTI-219 in DMS114 (FIG. 50A) and SHP77 (FIG. 49B) cells;Asterisks (*) denote significant differences (p<0.01) between controland COTI-219 treated cells;

FIG. 50 shows apoptotic cascade activation by COTI-219 as determined byrelative expression of cleaved caspase 3 and 9 using western blotanalysis following 12 h incubation with various concentrations ofCOTI-219;

FIGS. 51A and 51B show cell cycle analysis of DMS114 cells by FACSfollowing incubation for 24 h with indicated concentrations of COTI-219;duplicate experiments are shown; M1, Go/G1; M2, S/G; and

FIGS. 52A-B show a semi-log plot of the mean plasma concentration versustime following oral (FIG. 52A) and IV dosing (FIG. 52B).

FIG. 53 shows the effect of treatment on tumor weight. Tumor weightswere graphed as means (±SEM). The plus sign (+) indicates astatistically significant difference in tumor weight of the vehicle onlygroup compared to each of the combination & single agents on the sameday (P<0.05). The asterix (*) indicates a statistically significantdifference in tumor weight of the paclitaxel (Taxol®) only groupcompared to the COTI-2 and paclitaxel (Taxol®) combination agent groupson the same day (P<0.05). Statistical significance was determined usinga Student's T test.

FIG. 54 shows the effect of treatment on tumor weight. Tumor weights aregraphed as means (±SE). The asterix (*) indicates a statisticallysignificant difference in tumor weight of COTI-2 and Doxil® combinationagent groups relative to the vehicle only control group on the same day(P<0.05). The plus sign (+) indicates statistically significantdifference in tumor weight of the Doxil® single agent group versus thevehicle only control group on the same day (P<0.05). Statisticalsignificance was determined using a Student's T test.

FIG. 55 shows the effect of treatment on tumor weight. Tumor weights aregraphed as means (±SE). The asterix (*) indicates a statisticallysignificant difference in tumor weight of the COTI-2 (25 mg/kg) andErbitux® (1 mg/dose) combination agent group relative to the vehicleonly control group on the same day (P<0.05). The plus sign (+) indicatesa statistically significant difference in tumor weight of the COTI-2(12.5 mg/kg) and Erbitux® (1 mg/dose) combination agent group versus thevehicle only control group on the same day (P<0.05). Statisticalsignificance was determined using a Student's T test.

FIG. 56 shows the effect of treatment on tumor weight. Tumor weights aregraphed as means (±SE). Statistical significance was determined using aStudent's T test.

DETAILED DESCRIPTION Definitions

When describing the compounds, compositions, methods and uses of thisinvention, the following terms have the following meanings, unlessotherwise indicated.

The term “therapeutically effective amount” as used herein means thatamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue, system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician.

The compounds of the present invention may have asymmetric centers,chiral axes, and chiral planes (as described, for example, in: E. L.Eliel and S. H. Wilen, Stereo-chemistry of Carbon Compounds, John Wiley& Sons, New York, 1994, pages 1119-1190), and occur as racemates,racemic mixtures, and as individual diastereomers, with all possibleisomers and mixtures thereof, including optical isomers, being includedin the present invention. In addition, the compounds disclosed hereinmay exist as tautomers and both tautomeric forms are intended to beencompassed by the scope of the invention, even though only onetautomeric structure may be depicted.

Generally, reference to a certain element such as hydrogen or H is meantto, if appropriate, include all isotopes of that element.

Where the term “alkyl group” is used, either alone or within other termssuch as “haloalkyl group” and “alkylamino group”, it encompasses linearor branched carbon radicals having, for example, one to about twentycarbon atoms or, in specific embodiments, one to about twelve carbonatoms. In other embodiments, alkyl groups are “lower alkyl” groupshaving one to about six carbon atoms. Examples of such groups include,but are not limited thereto, methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl andthe like. In more specific embodiments, lower alkyl groups have one tofour carbon atoms.

The term “alkenyl group” encompasses linear or branched carbon radicalshaving at least one carbon-carbon double bond. The term “alkenyl group”can encompass conjugated and non-conjugated carbon-carbon double bondsor combinations thereof. An alkenyl group, for example and without beinglimited thereto, can encompass two to about twenty carbon atoms or, in aparticular embodiment, two to about twelve carbon atoms. In embodiments,alkenyl groups are “lower alkenyl” groups having two to about fourcarbon atoms. Examples of alkenyl groups include, but are not limitedthereto, ethenyl, propenyl, allyl, propenyl, butenyl and4-methylbutenyl. The terms “alkenyl group” and “lower alkenyl group”,encompass groups having “cis” and “trans” orientations, oralternatively, “E” and “Z” orientations.

The term “alkynyl group” denotes linear or branched carbon radicalshaving at least one carbon-carbon triple bond. The term “alkynyl group”can encompass conjugated and non-conjugated carbon-carbon triple bondsor combinations thereof. Alkynyl group, for example and without beinglimited thereto, can encompass two to about twenty carbon atoms or, in aparticular embodiment, two to about twelve carbon atoms. In embodiments,alkynyl groups are “lower alkynyl” groups having two to about ten carbonatoms. Some examples are lower alkynyl groups having two to about fourcarbon atoms. Examples of such groups include propargyl, butynyl, andthe like.

The term “halo” means halogens such as fluorine, chlorine, bromine oriodine atoms.

The term “haloalkyl group” encompasses groups wherein any one or more ofthe alkyl carbon atoms is substituted with halo as defined above.Specifically encompassed are monohaloalkyl, dihaloalkyl andpolyhaloalkyl groups including perhaloalkyl. A monohaloalkyl group, forone example, may have either an iodo, bromo, chloro or fluoro atomwithin the group. Dihalo and polyhaloalkyl groups may have two or moreof the same halo atoms or a combination of different halo groups. “Lowerhaloalkyl group” encompasses groups having 1-6 carbon atoms. In someembodiments, lower haloalkyl groups have one to three carbon atoms.Examples of haloalkyl groups include fluoromethyl, difluoromethyl,trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl,pentafluoroethyl, heptafluoropropyl, difluorochloromethyl,dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl anddichloropropyl.

The term “hydroxyalkyl group” encompasses linear or branched alkylgroups having, for example and without being limited thereto, one toabout ten carbon atoms, any one of which may be substituted with one ormore hydroxyl groups. In embodiments, hydroxyalkyl groups are “lowerhydroxyalkyl” groups having one to six carbon atoms and one or morehydroxyl groups. Examples of such groups include hydroxymethyl,hydroxyethyl, hydroxypropyl, hydroxybutyl and hydroxyhexyl.

The term “alkoxy group” encompasses linear or branched oxy-containinggroups each having alkyl portions of, for example and without beinglimited thereto, one to about ten carbon atoms. In embodiments, alkoxygroups are “lower alkoxy” groups having one to six carbon atoms.Examples of such groups include methoxy, ethoxy, propoxy, butoxy andtert-butoxy. In certain embodiments, lower alkoxy groups have one tothree carbon atoms. The “alkoxy” groups may be further substituted withone or more halo atoms, such as fluoro, chloro or bromo, to provide“haloalkoxy” groups. In other embodiments, lower haloalkoxy groups haveone to three carbon atoms. Examples of such groups includefluoromethoxy, chloromethoxy, trifluoromethoxy, trifluoroethoxy,fluoroethoxy, and fluoropropoxy.

The term “aromatic group” or “aryl group” means an aromatic group havingone or more rings wherein such rings may be attached together in apendent manner or may be fused. In particular embodiments, an aromaticgroup is one, two or three rings. Monocyclic aromatic groups may contain4 to 10 carbon atoms, typically 4 to 7 carbon atoms, and more typically4 to 6 carbon atoms in the ring. Typical polycyclic aromatic groups havetwo or three rings. Polycyclic aromatic groups having two ringstypically have 8 to 12 carbon atoms, preferably 8 to 10 carbon atoms inthe rings. Examples of aromatic groups include, but are not limited to,phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl,anthryl or acenaphthyl.

The term “heteroatom” means an atom other than carbon. Typically,heteroatoms are selected from the group consisting of sulfur,phosphorous, nitrogen and oxygen atoms. Groups containing more than oneheteroatom may contain different heteroatoms.

The term “heteroaromatic group” or “heteroaryl group” means an aromaticgroup having one or more rings wherein such rings may be attachedtogether in a pendent manner or may be fused, wherein the aromatic grouphas at least one heteroatom. Monocyclic heteroaromatic groups maycontain 4 to 10 member atoms, typically 4 to 7 member atoms, and moretypically 4 to 6 member atoms in the ring. Typical polycyclicheteroaromatic groups have two or three rings. Polycyclic aromaticgroups having two rings typically have 8 to 12 member atoms, moretypically 8 to 10 member atoms in the rings. Examples of heteroaromaticgroups include, but are not limited thereto, pyrrole, imidazole,thiazole, oxazole, furan, thiophene, triazole, pyrazole, isoxazole,isothiazole, pyridine, pyrazine, pyridazine, pyrimidine, triazine,indole, benzofuran, benzothiophene, benzimidazole, benzthiazole,quinoline, isoquinoline, quinazoline, quinoxaline and the like.

The term “carbocyclic group” means a saturated or unsaturatedcarbocyclic hydrocarbon ring. Carbocyclic groups are not aromatic.Carbocyclic groups are monocyclic or polycyclic. Polycyclic carbocyclicgroups can be fused, spiro, or bridged ring systems. Monocycliccarbocyclic groups may contain 4 to 10 carbon atoms, typically 4 to 7carbon atoms, and more typically 5 to 6 carbon atoms in the ring.Bicyclic carbocyclic groups may contain 8 to 12 carbon atoms, typically9 to 10 carbon atoms in the rings.

The term “heterocyclic group” means a saturated or unsaturated ringstructure containing carbon atoms and 1 or more heteroatoms in the ring.Heterocyclic groups are not aromatic. Heterocyclic groups are monocyclicor polycyclic. Polycyclic heterocyclic groups can be fused, spiro, orbridged ring systems. Monocyclic heterocyclic groups may contain 4 to 10member atoms (i.e., including both carbon atoms and at least 1heteroatom), typically 4 to 7, and more typically 5 to 6 in the ring.Bicyclic heterocyclic groups may contain 8 to 18 member atoms, typically9 or 10 member atoms in the rings. Representative heterocyclic groupsinclude, by way of example, pyrrolidine, imidazolidine, pyrazolidine,piperidine, 1,4-dioxane, morpholine, thiomorpholine, piperazine,3-pyrroline and the like.

The term “heterogeneous group” means a saturated or unsaturated chain ofnon-hydrogen member atoms comprising carbon atoms and at least oneheteroatom. Heterogeneous groups typically have 1 to 25 member atoms.More typically, the chain contains 1 to 12 member atoms, 1 to 10, andmost typically 1 to 6. The chain may be linear or branched. Typicalbranched heterogeneous groups have one or two branches, more typicallyone branch. Typically, heterogeneous groups are saturated. Unsaturatedheterogeneous groups may have one or more double bonds, one or moretriple bonds, or both. Typical unsaturated heterogeneous groups have oneor two double bonds or one triple bond. More typically, the unsaturatedheterogeneous group has one double bond.

The term “hydrocarbon group” or “hydrocarbyl group” means a chain of 1to 25 carbon atoms, typically 1 to 12 carbon atoms, more typically 1 to10 carbon atoms, and most typically 1 to 8 carbon atoms. Hydrocarbongroups may have a linear or branched chain structure. Typicalhydrocarbon groups have one or two branches, typically one branch.Typically, hydrocarbon groups are saturated. Unsaturated hydrocarbongroups may have one or more double bonds, one or more triple bonds, orcombinations thereof. Typical unsaturated hydrocarbon groups have one ortwo double bonds or one triple bond; more typically unsaturatedhydrocarbon groups have one double bond.

When the term “unsaturated” is used in conjunction with any group, thegroup may be fully unsaturated or partially unsaturated. However, whenthe term “unsaturated” is used in conjunction with a specific groupdefined herein, the term maintains the limitations of that specificgroup. For example, an unsaturated “carbocyclic group”, based on thelimitations of the “carbocyclic group” as defined herein, does notencompass an aromatic group.

The terms “carboxy group” or “carboxyl group”, whether used alone orwith other terms, such as “carboxyalkyl group”, denotes —(C═O)—O—.

The term “carbonyl group”, whether used alone or with other terms, suchas “aminocarbonyl group”, denotes —(C═O)—.

The terms “alkylcarbonyl group” denotes carbonyl groups which have beensubstituted with an alkyl group. In certain embodiments, “loweralkylcarbonyl group” has lower alkyl group as described above attachedto a carbonyl group.

The term “aminoalkyl group” encompasses linear or branched alkyl groupshaving one to about ten carbon atoms any one of which may be substitutedwith one or more amino groups. In some embodiments, the aminoalkylgroups are “lower aminoalkyl” groups having one to six carbon atoms andone or more amino groups. Examples of such groups include aminomethyl,aminoethyl, aminopropyl, aminobutyl and aminohexyl.

The term “alkylaminoalkyl group” encompasses aminoalkyl groups havingthe nitrogen atom independently substituted with an alkyl group. Incertain embodiments, the alkylaminoalkyl groups are“loweralkylaminoalkyl” groups having alkyl groups of one to six carbonatoms. In other embodiments, the lower alkylaminoalkyl groups have alkylgroups of one to three carbon atoms. Suitable alkylaminoalkyl groups maybe mono or dialkyl substituted, such as N-methylaminomethyl,N,N-dimethyl-aminoethyl, N,N-diethylaminomethyl and the like.

The term “aralkyl group” encompasses aryl-substituted alkyl groups. Inembodiments, the aralkyl groups are “lower aralkyl” groups having arylgroups attached to alkyl groups having one to six carbon atoms. In otherembodiments, the lower aralkyl groups phenyl is attached to alkylportions having one to three carbon atoms. Examples of such groupsinclude benzyl, diphenylmethyl and phenylethyl. The aryl in said aralkylmay be additionally substituted with halo, alkyl, alkoxy, haloalkyl andhaloalkoxy.

The term “arylalkenyl group” encompasses aryl-substituted alkenylgroups. In embodiments, the arylalkenyl groups are “lower arylalkenyl”groups having aryl groups attached to alkenyl groups having two to sixcarbon atoms. Examples of such groups include phenylethenyl. The aryl insaid arylalkenyl may be additionally substituted with halo, alkyl,alkoxy, haloalkyl and haloalkoxy.

The term “arylalkynyl group” encompasses aryl-substituted alkynylgroups. In embodiments, arylalkynyl groups are “lower arylalkynyl”groups having aryl groups attached to alkynyl groups having two to sixcarbon atoms. Examples of such groups include phenylethynyl. The aryl insaid aralkyl may be additionally substituted with halo, alkyl, alkoxy,haloalkyl and haloalkoxy. The terms benzyl and phenylmethyl areinterchangeable.

The term “alkylthio group” encompasses groups containing a linear orbranched alkyl group, of one to ten carbon atoms, attached to a divalentsulfur atom. In certain embodiments, the lower alkylthio groups have oneto three carbon atoms. An example of “alkylthio” is methylthio, (CH₃S—).

The term “alkylamino group” denotes amino groups which have beensubstituted with one alkyl group and with two alkyl groups, includingterms “N-alkylamino” and “N,N-dialkylamino”. In embodiments, alkylaminogroups are “lower alkylamino” groups having one or two alkyl groups ofone to six carbon atoms, attached to a nitrogen atom. In otherembodiments, lower alkylamino groups have one to three carbon atoms.Suitable “alkylamino” groups may be mono or dialkylamino such asN-methylamino, N-ethylamino, N,N-dimethylamino, N,N-diethylamino and thelike.

The term “arylamino group” denotes amino groups which have beensubstituted with one or two aryl groups, such as N-phenylamino. The“arylamino” groups may be further substituted on the aryl ring portionof the group.

The term “heteroarylamino” denotes amino groups which have beensubstituted with one or two heteroaryl groups, such as N-thienylamino.The “heteroarylamino” groups may be further substituted on theheteroaryl ring portion of the group.

The term “aralkylamino group” denotes amino groups which have beensubstituted with one or two aralkyl groups. In other embodiments, thereare phenyl-C₁-C₃-alkylamino groups, such as N-benzylamino. The“aralkylamino” groups may be further substituted on the aryl ringportion of the group.

The term “alkylaminoalkylamino group” denotes alkylamino groups whichhave been substituted with one or two alkylamino groups. In embodiments,there are C₁-C₃-alkylamino-C₁-C₃-alkylamino groups.

The term “arylthio group” encompasses aryl groups of six to ten carbonatoms, attached to a divalent sulfur atom. An example of “arylthio” isphenylthio. The term “aralkylthio group” encompasses aralkyl groups asdescribed above, attached to a divalent sulfur atom. In certainembodiments there are phenyl-C₁-C₃-alkylthio groups. An example of“aralkylthio” is benzylthio.

The term “aryloxy group” encompasses optionally substituted aryl groups,as defined above, attached to an oxygen atom. Examples of such groupsinclude phenoxy.

The term “aralkoxy group” encompasses oxy-containing aralkyl groupsattached through an oxygen atom to other groups. In certain embodiments,aralkoxy groups are “lower aralkoxy” groups having optionallysubstituted phenyl groups attached to lower alkoxy group as describedabove.

The term “cycloalkyl group” includes saturated carbocyclic groups. Incertain embodiments, cycloalkyl groups include C₃-C₆ rings. Inembodiments, there are compounds that include, cyclopentyl, cyclopropyl,and cyclohexyl.

The term “cycloalkenyl group” includes carbocyclic groups that have oneor more carbon-carbon double bonds; conjugated or non-conjugated, or acombination thereof. “Cycloalkenyl” and “cycloalkyldienyl” compounds areincluded in the term “cycloalkenyl”. In certain embodiments,cycloalkenyl groups include C₃-C₆ rings. Examples include cyclopentenyl,cyclopentadienyl, cyclohexenyl and cycloheptadienyl. The “cycloalkenyl”group may have 1 to 3 substituents such as lower alkyl, hydroxyl, halo,haloalkyl, nitro, cyano, alkoxy, lower alkylamino, and the like.

The term “suitable substituent”, “substituent” or “substituted” used inconjunction with the groups described herein refers to a chemically andpharmaceutically acceptable group, i.e., a moiety that does not negatethe therapeutic activity of the inventive compounds. It is understoodthat substituents and substitution patterns on the compounds of theinvention may be selected by one of ordinary skill in the art to providecompounds that are chemically stable and that can be readily synthesizedby techniques known in the art, as well as those methods set forthbelow. If a substituent is itself substituted with more than one group,it is understood that these multiple groups may be on the samecarbon/member atom or on different carbons/member atoms, as long as astable structure results. Illustrative examples of some suitablesubstituents include, cycloalkyl, heterocyclyl, hydroxyalkyl, benzyl,carbonyl, halo, haloalkyl, perfluoroalkyl, perfluoroalkoxy, alkyl,alkenyl, alkynyl, hydroxy, oxo, mercapto, alkylthio, alkoxy, aryl orheteroaryl, aryloxy or heteroaryloxy, aralkyl or heteroaralkyl, aralkoxyor heteroaralkoxy, HO—(C═O)—, amido, amino, alkyl- and dialkylamino,cyano, nitro, carbamoyl, alkylcarbonyl, alkoxycarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, arylcarbonyl, aryloxycarbonyl,alkylsulfonyl, and arylsulfonyl. Typical substituents include aromaticgroups, substituted aromatic groups, hydrocarbon groups including alkylgroups such as methyl groups, substituted hydrocarbon groups such asbenzyl, and heterogeneous groups including alkoxy groups such as methoxygroups.

The term “fused” means in which two or more carbons/member atoms arecommon to two adjoining rings, e.g., the rings are “fused rings”.

The pharmaceutically acceptable salts of the compounds of this inventioninclude the conventional non-toxic salts of the compounds of thisinvention as formed, e.g., from non-toxic inorganic or organic acids.For example, such conventional non-toxic salts include those derivedfrom inorganic acids such as hydrochloric, hydrobromic, sulfuric,sulfamic, phosphoric, nitric and the like; and the salts prepared fromorganic acids such as acetic, propionic, succinic, glycolic, stearic,lactic, malic, tartaric, citric, ascorbic, pamoic, maleic,hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic,2-acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, trifluoroacetic and the like. Preferredsalts include oxalate and tartrate salts.

The pharmaceutically acceptable salts of the compounds of this inventioncan be synthesized from the compounds of this invention which contain abasic or acidic moiety by conventional chemical methods. Generally, thesalts of the basic compounds are prepared either by ion exchangechromatography or by reacting the free base with stoichiometric amountsor with an excess of the desired salt-forming inorganic or organic acidin a suitable solvent or various combinations of solvents. Similarly,the salts of the acidic compounds are formed by reactions with theappropriate inorganic or organic base.

The present invention includes pharmaceutically acceptable salts,solvates and prodrugs of the compounds of the invention and mixturesthereof.

The terms “comprising”, “having” and “including”, and various endingsthereof, are meant to be open ended, including the indicated componentbut not excluding other elements.

Therapeutically active compounds of the present invention comprisethiosemicarbazones, in particular thiosemicarbazones represented byFormula I:

wherein:R₁ and R₂ together form a substituted or unsubstituted polycyclic ring.The ring has at least two ring systems. The two ring systems have afirst ring system that is bonded to C1 and a second ring system that isfused to the first ring system.

In one embodiment, the first ring system is a substituted orunsubstituted aromatic group and the second ring system is a substitutedor unsubstituted aromatic group, a substituted or unsubstitutedheteroaromatic group, a substituted or unsubstituted carbocyclic group,or a substituted or unsubstituted heterocyclic group.

In a second embodiment, the first ring system is a substituted orunsubstituted heteroaromatic group and the second ring system is asubstituted or unsubstituted aromatic group, a substituted orunsubstituted heteroaromatic group, a substituted or unsubstitutedcarbocyclic group, a substituted or unsubstituted heterocyclic group.

In a further embodiment, the first ring system is a substituted orunsubstituted saturated carbocyclic group and the second ring system isa substituted or unsubstituted aromatic group, a substituted orunsubstituted unsaturated carbocyclic group, a substituted orunsubstituted heterocyclic group, or a substituted or unsubstituted ringB:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom.

In another embodiment, the first ring system is a substituted orunsubstituted unsaturated carbocyclic group and the second ring systemis a substituted or unsubstituted aromatic group, a substituted orunsubstituted carbocyclic group, a substituted or unsubstitutedheterocyclic group, or a substituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom.

In yet another embodiment, the first ring system is a substituted orunsubstituted heterocyclic group, the second ring system is asubstituted or unsubstituted heteroaromatic group, a substituted orunsubstituted carbocyclic group, or a substituted or unsubstitutedheterocyclic group.

In another embodiment relating to the above-identified embodiments, thefirst ring system is a five- or six-membered ring.

In embodiments, the R₃ to R₁₁ groups are each independently selectedfrom H, a substituted or unsubstituted hydrocarbon group, a substitutedor unsubstituted heterogeneous group, a substituted or unsubstitutedcarbocyclic group, a substituted or unsubstituted heterocyclic group,substituted or unsubstituted aromatic, or a substituted or unsubstitutedheteroaromatic. The R₁₂ group is selected from H or a hydrocarbyl groupand Y is selected from a heteroatom or a carbon atom. “A” is selectedfrom a substituted or unsubstituted hydrocarbon group, a substituted orunsubstituted heterogeneous group, a substituted or unsubstitutedcarbocyclic group, a substituted or unsubstituted heterocyclic group,substituted or unsubstituted aromatic, or a substituted or unsubstitutedheteroaromatic and “n” is an integer.

Therapeutically active compounds described herein can be thethiosemicarbazone compounds of Formula I, pharmaceutically-acceptablesalts thereof, hydrates thereof, solvates thereof, tautomers thereof,optical isomers thereof, or a combination thereof.

In a specific embodiment, the first ring system of the compound ofFormula I is a substituted or unsubstituted carbocyclic group and thesecond ring system is a substituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom. In a more specific embodiment, ring B is a pyridine ring,typically fused to the first ring at C2 and C3 of the pyridine ring.

Although a first and second ring system is described herein, thesubstituted or unsubstituted polycyclic ring may further comprise otherring systems other than the first and second ring systems. For example,a third ring system may also be fused to the first ring system. Thethird ring system can be, for instance, a substituted or unsubstitutedaromatic group, a substituted or unsubstituted heteroaromatic group, asubstituted or unsubstituted carbocyclic group, or a substituted orunsubstituted heterocyclic group. Typically, the third ring system is asubstituted or unsubstituted heteroaromatic group or a substituted orunsubstituted heterocyclic group.

With respect to the embodiments described above with respect to FormulaI, typically “n” is 0 or 1. If “n” is 1, “A” is typically a substitutedor unsubstituted heteroaromatic group, in particular, a pyridinyl group.

Also, with respect to the embodiments of Formula I, Y is typically anitrogen atom. The ring:

can be a variety of rings. The ring can be a substituted orunsubstituted thiomorpholinyl group, a substituted or unsubstitutedmorpholinyl group, a substituted or unsubstituted piperidinyl group, ora substituted or unsubstituted piperazinyl group.

In specific embodiments of Formula I, R₇ is a substituted orunsubstituted alkyl group or a substituted or unsubstitutedheteroaromatic group and R₃ to R₆ and R₈ to R₁₂ are each independentlyselected from H or a substituted or unsubstituted hydrocarbon group.More specifically, R₇ can be the substituted or unsubstituted alkylgroup or a substituted or unsubstituted pyridyl group and R₃ to R₆ andR₈ to R₁₂ are each H.

In specific embodiments, the compound of Formula I can be:

Such compounds may be used and/or in the form of apharmaceutically-acceptable salt, hydrate, solvate or any combinationthereof.

The compounds of Formula I described herein can be prepared as follows:

a) reacting a compound of Formula II:

with a compound of Formula IIA:

to form an intermediate of Formula III:

b) reacting the Intermediate of Formula III with R₁₂NHNH₂ to form anIntermediate of Formula IV:

c) reacting the Intermediate of Formula IV with a ketone:

under condensation conditions, to form the compound of Formula I. Inspecific embodiments, the above-identified synthetic method can be usedwhen “n” is 0 or 1; more typically, when “n” is 0.

The compounds of Formula I described herein can also be prepared asfollows:

a) dithioesterifying a halo compound of Formula V:

to form an intermediate of Formula VI, wherein R, R′₁ or R′₂ issubstituted or unsubstituted hydrocarbon group, a substituted orunsubstituted heterogeneous group, a substituted or unsubstitutedcarbocyclic group, a substituted or unsubstituted heterocyclic group,substituted or unsubstituted aromatic, or a substituted or unsubstitutedheteroaromatic:

b) reacting the Intermediate of Formula VI with R₁₂NHNH₂ to form anIntermediate of Formula IV:

c) reacting the Intermediate of Formula IV with a ketone:

under condensation conditions, to form the compound of Formula I. Inspecific embodiments, the above-identified synthetic method can be usedwhen “n” is 0 or 1; more typically, when “n” is 1.

The compounds of Formula I described herein can also be prepared asfollows:

a) esterifying compound of Formula IIA:

to form an intermediate of Formula VII, wherein R is substituted orunsubstituted hydrocarbon group, a substituted or unsubstitutedheterogeneous group, a substituted or unsubstituted carbocyclic group, asubstituted or unsubstituted heterocyclic group, substituted orunsubstituted aromatic, or a substituted or unsubstitutedheteroaromatic:

b) reacting the Intermediate of Formula VII with R₁₂NHNH₂ to form anIntermediate of Formula VIII:

c) reacting the Intermediate of Formula VIII with a thiation agent toform an Intermediate of Formula IV:

c) reacting the Intermediate of Formula IV with a ketone:

under condensation conditions, to form the compound of Formula I.Examples of a thiation agent include, but are not limited to, phosphoruspentasulfide or Lawesson's reagent. In specific embodiments, theabove-identified synthetic method can be used when “n” is 0 or 1; moretypically, when “n” is 1.

The compounds described herein are useful in the treatment of cancer.High levels of activity for in vitro and in vivo testing have beenobserved against cancers and cancer models using the compounds of thepresent invention. This may lead to reduced dosages as compared withconventional therapeutic dosages of known agents.

The cancer treated may be, for example, lung cancer, cervical cancer,ovarian cancer, cancer of CNS, skin cancer, prostate cancer, sarcoma,breast cancer, leukemia, colorectal cancer, endometrial cancer, headcancer, neck cancer or kidney cancer. More typically, the cancer may besmall cell lung cancer, breast cancer, acute leukemia, chronic leukemia,colorectal cancer, ovarian cancer, endometrial cancer or brain cancer.The cancer may be a carcinoma. The carcinoma may be selected from smallcell carcinomas, cervical carcinomas, glioma, astrocytoma, prostatecarcinomas, ovarian carcinomas, melanoma, breast carcinomas, endometrialcarcinomas or colorectal carcinomas. Compounds of the present inventionmay be even more particularly useful in the treatment of small cell lungcancer (SCLC) carcinomas.

Compounds useful according to the present invention can have an IC₅₀ fora cancer cell population when administered as single agents of less thanabout 1000 nM. In specific embodiments, compounds of the presentinvention show efficacy against SHP77 cells when administered as singleagents at IC50's of less than about 1000 nM, typically less than about800 nM, more typically less than about 500 nM, even more typically lessthan about 200 nM.

Compounds useful according to the present invention show efficacyagainst DMS144 cells when administered as single agents at IC₅₀'s ofless than about 1000 nM, typically less than about 750 nM, moretypically less than about 500 nM, even more typically less than about300 nM, yet more typically less than about 100 nM.

Compounds useful according to the present invention show efficacyagainst U87 cells when administered as single agents at IC50's of lessthan about 2500 nM, typically less than about 1000 nM, more typicallyless than about 480 nM, even more typically less than about 200 nM, yetmore typically less than about 75 nM.

Compounds useful according to the present invention show efficacyagainst SNB-19 cells when administered as single agents at IC50's ofless than about 2150 nM, typically less than about 1500 nM, moretypically less than about 800 nM, even more typically less than about100 nM, yet more typically less than about 50 nM, still more typicallyless than about 15 nM.

Compounds useful according to the present invention are effective inreducing the size of malignant human cancer tumors created from SHP77,DMS114, N417 and/or U87 cell lines.

Compounds useful according to the present invention can penetrate theblood brain barrier of a mammal, typically, a human.

Compounds useful according to the present invention may exhibit areduced tendency to induce cellular resistance to either their ownanti-cancer effects or the effects of other anti-cancer agents.Therefore, use of the compounds of the present invention to treat acancer, as single agents or as part of a combination, may inhibitdevelopment of a drug resistant form of that cancer. Without wishing tobe limited by theory, it is believed that the compounds of the presentinvention may inhibit development of P-glycoprotein mediated drugresistance and/or function as weak substrates of P-glycoprotein.

Certain compounds useful according to the present invention may exhibitreduced toxicity as compared with conventionally administered agents.

The compounds of this invention may be administered to mammals,typically humans, either alone or, in combination with pharmaceuticallyacceptable carriers or diluents, optionally with known adjuvants, suchas alum, in a pharmaceutical composition, according to standardpharmaceutical practice. The compounds can be administered orally orparenterally, including the intravenous, intramuscular, intraperitoneal,and subcutaneous routes of administration.

As noted, compounds useful according to the present invention may beadministered orally, unlike most current cancer therapies, which areadministered intravenously. For oral use of a compound or composition inthis invention, the selected compound may be administered, for example,in the form of tablets or capsules, or as an aqueous solution orsuspension. In the case of tablets for oral use, carriers which arecommonly used include lactose and corn starch, and lubricating agents,such as magnesium stearate, are commonly added. For oral administrationin capsule form, useful diluents include lactose and dried corn starch.When aqueous suspensions are required for oral use, the activeingredient is combined with emulsifying and suspending agents. Ifdesired, certain sweetening and/or flavoring agents may be added. Forintramuscular, intraperitoneal, subcutaneous and intravenous use,sterile solutions of the active ingredient are usually prepared, and thepH of the solutions should be suitably adjusted and buffered. Forintravenous use, the total concentration of solutes should be controlledin order to render the preparation isotonic.

At least about 50% of compounds useful according to the presentinvention can be orally absorbed by a mammal. In specific embodiments,at least about 60%; about 60% to about 85%; about 65%; about 70%; about72%; about 73%, about 75%; about 80%; about 82%; or about 85% of thecompound can be orally absorbed by a mammal, more typically, a human.“Oral absorption” is used in the context of how the compound/compositionis delivered and absorbed into the blood. Typically, thecompound/composition is administered orally and crosses a mucosalmembrane of the gastro-intestinal tract, typically in the intestines.However, other methods of contacting the compounds/compositions of thepresent invention with the mucosal membrane of the gastrointestinaltract may also be used.

The compounds of the present invention may also be combined and/orco-administered with other therapeutic agents that are selected fortheir particular usefulness against the cancer that is being treated.For example, the compounds of the present invention may be combinedand/or co-administered with anti-cancer agent(s).

Examples of anti-cancer agents include, without being limited thereto,the following: estrogen receptor modulators, androgen receptormodulators, retinoid receptor modulators, cytotoxic agents, mTORinhibitors, particularly mTOR-Raptor complex inhibitors,antiproliferative agents, tyrosine kinase inhibitors, prenyl-proteintransferase inhibitors, HMG-CoA reductase inhibitors, HIV proteaseinhibitors, reverse transcriptase inhibitors, other angiogenesisinhibitors and combinations thereof. The present compounds may also beuseful with other therapies such as when co-administered with radiationtherapy.

“Estrogen receptor modulators” refers to compounds which interfere orinhibit the binding of estrogen to the receptor, regardless ofmechanism. Examples of estrogen receptor modulators include, but are notlimited thereto, tamoxifen, raloxifene, idoxifene, LY353381, LY117081,toremifene, fulvestrant,4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate,4,4′-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646.

“Androgen receptor modulators” refers to compounds which interfere orinhibit the binding of androgens to the receptor, regardless ofmechanism. Examples of androgen receptor modulators include finasterideand other 5α-reductase inhibitors, nilutamide, flutamide, bicalutamide,liarozole, and abiraterone acetate.

“Retinoid receptor modulators” refers to compounds which interfere orinhibit the binding of retinoids to the receptor, regardless ofmechanism. Examples of such retinoid receptor modulators includebexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid,α-difluoromethylomithine, ILX23-7553, trans-N-(4′-hydroxyphenyl)retinamide and N-4-carboxyphenyl retinamide.

“Cytotoxic agents” refer to compounds which cause cell death primarilyby interfering directly with the cell's functioning or inhibit orinterfere with cell myosis, including alkylating agents, tumor necrosisfactors, intercalators, microtubulin inhibitors, and topoisomeraseinhibitors.

Examples of cytotoxic agents include, but are not limited thereto,cyclophosphamide ifosfamide, hexamethylmelamine, tirapazimine, sertenef,cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, mitomycin,altretamine, prednimustine, dibromodulcitol, ranimustine, fotemustine,nedaplatin, oxaliplatin, temozolomide, doxorubicin (Doxil™),heptaplatin, estramustine, improsulfan tosilate, trofosfamide,nimustine, dibrospidium chloride, pumitepa, lobaplatin, satraplatin,profiromycin, cisplatin, irofulven, dexifosfamide,cis-aminedichloro(2-methyl-pyridine) platinum, benzylguanine,glufosfamide, GPX100, (trans, trans,trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(chloro)-platinum(II)]tetrachloride, diarizidinylspermine, arsenic trioxide,1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin,idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin,pinafide, valrubicin, amrubicin, antineoplaston,3′-deamino-3′-morpholino-13-deoxo-10-hydroxycaminomycin, annamycin,galarubicin, elinafide, MEN10755, and4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunor-ubicin (seeInternational Patent Application No. WO 00/50032).

“mTOR inhibitors” are a subset of the cytotoxic agents and referparticularly to inhibitors of the mTOR-Raptor complex. Included in thedefinition of mTOR inhibitors are anti-cancer agents such as rapamycinand its derivatives, sirolimus, temsirolimus, everolimus, zotarolimusand deforolimus.

Examples of microtubulin inhibitors include paclitaxel (Taxol®),vindesine sulfate, 3′,4′-didehydro-4′-deoxy-8′-norvincaleukoblastine,docetaxel, rhizoxin, dolastatin, mivobulin isethionate, auristatin,cemadotin, RPR109881, BMS184476, vinflunine, cryptophycin,2,3,4,5,6-pentafluoro-N-(−3-fluoro-4-methoxyphenyl)benzene sulfonamide,anhydrovinblastine,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide,TDX258, and BMS188797.

Some examples of topoisomerase inhibitors are topotecan, hycaptamine,irinotecan, rubitecan,6-ethoxypropionyl-3′,4′-O-exo-benzylidene-chartreusin,9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H)propanamine,1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1-1H,12Hbenzo[de]pyrano[3′,4′:b,7]indolizino[1,2b]quinoline-10,13(9H,15H)dione,lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350,BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane,2′-dimethylamino-2′-deoxy-etoposide, GL331,N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazo-le-1-carboxamide,asulacrine,(5a,5aB,8aa,9b)-9-[2-[N-[2-(dimethylamino)-ethyl]-N-methylamino]ethyl]-5-[4-Hydroxy-3,5-dimethoxyphenyl]-5,5a,6,8,8a,-9-hexohydrofuro(3′,4′:6,7)naphtho(2,3-d)-1,3-dioxol-6-one,2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridiniu-m,6,9-bis[(2-aminoethyl)amino]benzo[g]isoquinoline-5,10-dione,5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-py-razolo[4,5,1-de]acridin-6-one,N-[1-[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide,N-(2-(dimethylamino)ethyl)acrid-ine-4-carboxamide,6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2-,1-c]quinolin-7-one,and dimesna.

“Antiproliferative agents” includes BCNU, antisense RNA and DNAoligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001,and antimetabolites such as floxuridine, enocitabine, carmofur, tegafur,pentostatin, doxifluridine, trimetrexate, fludarabine, capecitabine,galocitabine, cytarabine ocfosfate, fosteabine sodium hydrate,raltitrexed, paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed,pemetrexed, neizarabine, 2′-deoxy-2′-methylidenecytidine,2′-fluoromethylene-2′-deoxy-cytidine,N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N′-(3,4-dichlorophenyl)urea,N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycer-o-B-L-manno-heptopyranosyl]adenine,aplidine, ecteinascidin, troxacitabine,4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamicacid, aminopterin, 5-fluorouracil, alanosine,11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-1,11-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-ylacetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase,2′-cyano-2′-deoxy-N4-palmitoyl-1-B-D-arabino furanosyl cytosine, and3-aminopyridine-2-carboxaldehyde thiosemicarbazone.

“Antiproliferative agents” also includes monoclonal antibodies to growthfactors, other than those listed under “angiogenesis inhibitors”, suchas trastuzumab, and tumor suppressor genes, such as p53, which can bedelivered via recombinant virus-mediated gene transfer (see U.S. Pat.No. 6,069,134, for example).

Some specific examples of tyrosine kinase inhibitors includeN-(trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide,3-[(2,4-dimethylpyrrol-5-yl)methylidenyl)indolin-2-one,17-(allylamino)-17-demethoxygeldanamycin,4-(3-chloro-4-fluorophenylamino-)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]-quinazoline,N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine,2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one,SH1382, genistein,4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethanesulfonate, 4-(3-bromo-4-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline,4-(4′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline,N-4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazinamine, and Tarceva®(erlotinib).

Typical examples of anti-cancer agents suitable for use in combinationtherapies or compositions according to the invention include cisplatin,rapamycin, tecrolimus, temsirolimus, paclitaxel, erlotinib, cetuximaband doxorubicin.

If formulated as a fixed dose, such combination products employ thecompounds of this invention within the dosage range described below andthe other pharmaceutically active agent(s) within its approved dosagerange. Compounds of the present invention may alternatively be usedsequentially with known pharmaceutically acceptable agent(s) when acombination formulation is inappropriate.

The term “administration” (e.g., “administering” a compound) inreference to a compound of the invention means introducing the compoundor a prodrug of the compound into the system of the animal in need oftreatment. When a compound of the invention or prodrug thereof isprovided in combination with one or more other active agents (e.g., acytotoxic agent, etc.), “administration” and its variants are eachunderstood to include concurrent and sequential introduction of thecompound or prodrug thereof and other agents.

The term “treating cancer” or “treatment of cancer” refers toadministration to a mammal afflicted with a cancerous condition andrefers to an effect that alleviates the cancerous condition by killingthe cancerous cells, but also to an effect that results in theinhibition of growth and/or metastasis of the cancer.

When a compound according to this invention is administered into a humansubject, the daily dosage will normally be determined by the prescribingphysician with the dosage generally varying according to the age,weight, and response of the individual patient, as well as the severityof the patient's symptoms.

In one exemplary application, a suitable amount of compound isadministered to a mammal undergoing treatment for cancer. Administrationoccurs in an amount from about 0.01 mg/kg of body weight to greater thanabout 100 mg/kg of body weight per day; from about 0.01 mg/kg of bodyweight to about 500 mg/kg of body weight per day; from about 0.01 mg/kgof body weight to about 250 mg/kg of body weight per day; or 0.01 mg/kgof body weight to about 100 mg/kg of body weight per day. These dosagescan be more particularly used orally.

The compounds of this invention may be prepared by employing reactionsand standard manipulations that are known in the literature orexemplified herein.

Without wishing to be limited by theory, it is believed that thetherapeutic compounds described herein, especially those compoundsaccording to Formula I, function by reducing or preventing activity ofAkt, particularly Akt2, via inhibition of Serine 473 (“Ser 473”)phosphorylation. Akt (also known as protein kinase B) is aserine/threonine kinase, which in mammals comprises three isoforms knownas Akt1, Akt2, and Akt3. Cancerous cells often auto-phosphorylate Aktand an interruption in this cycle is useful in preventing tumour growth.Inhibition of Ser 473 phosphorylation may decrease phosphorylation ofCaspase 9, which may have the effect of increasing active Caspase 9 andinducing apoptosis via Caspase 3.

Again, without wishing to be limited by theory, it is believed that theMamallian Targets of Rapamycin (mTOR) cell signaling pathway may beinvolved in a compound's ability to inhibit Akt phosphorylation at Ser473. The preponderance of the available evidence supports the contentionthat the mTOR-Rictor complex is the agent responsible for thephosphorylation event at Ser 473 of Akt. At present two major componentsof mTOR, namely mTOR-Raptor complex and mTOR-Rictor complex, have beenidentified. mTOR-Raptor complex is centrally involved in cellularimmunity and organ rejection. mTOR-Raptor complex inhibitors have alsodemonstrated both in vitro and in vivo anti-neoplastic properties.

Conventional mTOR inhibitors are thought to interfere with themTOR-Raptor complex. Prior to this disclosure, there were no recognizedtherapeutic agents shown to inhibit the mTOR-Rictor complex. Thecompounds described herein according to Formula I include examples ofmTOR-Rictor complex inhibitors, particularly the compound designated asCOTI-2. By inhibiting Ser 473 phosphorylation of Akt2, the compoundsdescribed herein implicate the mTOR-Rictor complex and therefore providea novel mechanistic pathway for anti-cancer therapy.

It has been shown herein that there is a synergistic effect, defined asa greater than additive benefit, provided by co-administration of anmTOR-Raptor complex inhibitor and an mTOR-Rictor complex inhibitor inthe treatment of cancer. It is believed that, by acting simultaneouslyon both the Raptor and Rictor components of mTOR, superior inhibition ofmTOR can be achieved leading to improved cancer treatment outcomes, atleast for cancers normally susceptible to treatment by conventional mTORinhibitors. In particular, those cancers that are treatable byinhibition of Akt and particularly Akt2, for example via inhibition ofSer473 phosphorylation, are believed to be especially good candidatesfor exhibiting synergistic improvements in treatment outcome whenexposed to co-administration of mTOR-Raptor and mTOR-Rictor complexinhibitors.

However, the synergistic improvement in treatment outcome is not onlylimited to combinations of the therapeutic compounds of the inventionand mTOR inhibitors. Synergy has also been observed with otheranti-cancer agents, for example the cytotoxic agents cisplatin,paclitaxel, erlotinib, cetuximab and doxorubicin. Therefore, there arenumerous anti-cancer agents that exhibit synergistic improvements intreatment outcome when co-administered with the therapeutic compounds ofthe invention, especially those according to Formula I.

These methods and uses are applicable to cancers characterized byexpression of Akt, particularly cancers characterized byauto-phosphorylation of Akt. Cancers wherein the mTOR cell signalingpathway has been implicated or where administration of mTOR-Raptorcomplex inhibitors are part of accepted treatment practices, forexample, small cell lung cancer, carcinoid tumors, metastatic colorectalcancer, islet cell carcinoma, metastatic renal cell carcinoma breastcancer, ovarian cancer or endometrial cancer, adenocarcinomas,glioblastoma multiforme, bronchoalveolar lung cancers, sarcomas(including leiomyosarcomas), non-Hodgkin's lymphoma, neuroendocrinetumors, and neuroblastoma are particularly suitable examples.

Although any combination of doses may be used, typically doses of theanti-cancer agent and/or the therapeutic compound that provide asynergistic effect, or greater than additive benefit, are used. Forexample, doses of the anti-cancer agent and/or the therapeutic compoundmay be selected to synergistically lower overall toxicity whilemaintaining substantially the same overall treatment effect on cancerouscells as observed when the anti-cancer agent is administered alone. Inanother example, doses of the anti-cancer agent and/or the therapeuticcompound may be selected to produce substantially the same overalltoxicity while synergistically increasing the treatment effect oncancerous cells as observed when the anti-cancer agent is administeredalone.

Due to this synergistic behaviour, anti-cancer agents, particularlymTOR-Raptor complex inhibitors, may be advantageously administered atdoses lower than currently approved doses by co-administration withtherapeutic compounds according to the invention without substantiallyreducing the efficacy of the cancer treatment. This has the benefit ofreducing toxicity of the combination. In addition, the toxicity of thetherapeutic compound being co-administered may be less due to either alower required dose or improved toxicological properties; this has theeffect of further lowering overall toxicity of the combination withoutcompromising the overall treatment effect.

The use of the therapeutic compound at a lower dose than the anti-canceragent includes doses that are, for example, at most about 80%, about50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 3%,about 2%, about 1%, about 0.75%, about 0.5%, about 0.25%, or about 0.1%,etc. of the dose of the anti-cancer compound. At these doses, asynergistic effect in the treatment of cancerous cells may be observed.

Without wishing to be limited by theory, compounds described herein mayalso function by interrupting cellular metabolic cycles in whichp-glycoprotein (Pgp) participates. These cellular cycles are known to“pump” cellular toxins such as some chemotherapeutic agents across thecellular membrane out of the cell and serve to lower the concentrationof therapeutic agents within cancerous cells. By interrupting thisprocess, the compounds described herein are allowed to accumulate tohigher concentrations within cancerous cells, thereby providing themwith substantially increased efficacy in the treatment of cancer. Thecompounds are characterized in that they inhibit p-glycoprotein and/orare weak substrates for p-glycoprotein.

The effect on P-glycoprotein of compounds described herein makes themparticularly suitable for use in combination therapy with otheranti-cancer agents, in particular cytotoxic agents such as cisplatin andpaclitaxel (Taxol™), since these agents are thereby allowed toaccumulate to higher levels within the cancerous cells. A synergisticeffect or greater than additive benefit may therefore be observed whencompounds described herein are administered in combination with otheranti-cancer agents, in particular cytotoxic agents such as cisplatin andpaclitaxel (see FIGS. 19-22).

Also provided herein is a therapeutic method or use for the treatment ofcancer comprising potentiating an anti-cancer agent, particularly acytotoxic agent, by co-administering a therapeutic compound as describedherein that is a p-glycoprotein inhibitor and/or weak p-glycoproteinsubstrate, preferably in a manner producing a synergistic therapeuticeffect.

Without wishing to be limited by theory, compounds described herein maywork by preventing activity of Akt via inhibition of Ser 473phosphorylation, and also by interrupting cellular metabolic cycles inwhich P-glycoprotein (Pgp) participates This defines a further novelsub-class of therapeutic compounds; compounds that are both Ser 473phosphorylation inhibitors and P-glycoprotein inhibitors. Althoughapplicable to a wide variety of cancers, this novel sub-class oftherapeutic compounds is particularly useful in the treatment of cancerswherein administration of mTOR-Raptor complex inhibitors and/orcytotoxic agents is/are part of accepted treatment practices.

The synergistic improvement in treatment outcome may be a greater thanadditive improvement in overall efficacy obtained by co-administrationas compared with administration of either agent alone. This may beillustrated through obtaining a “combination index” (CI) value of lessthan 1, as is described in detail in Example 18, hereinafter.

It has also been shown that therapeutic compounds according to Formula Iexhibit improved efficacy in treatment of cancers characterized byover-expression of RAS, by an EGFR mutation, and/or by over-expressionof AKT2. Such compounds are beneficial as many anti-cancer agents areknown to have difficulty in obtaining efficacy in the treatment of thesetypes of cancers. In particular, compounds of Formula I have been shownefficacious in treatment of cancers characterized by the KRAS mutation.Exemplary forms of this cancer include leukemia, colon cancer,colorectal cancer, pancreatic cancer, lung cancer, multiple myeloma,endometrial cancer, and ovarian cancer. Cancers characterized by theEGFR mutation include lung cancer, glioblastoma, colon cancer, gastriccancer, renal cancer, prostate cancer, breast cancer, and ovariancancer. In particular, compounds according to formula I have shownefficacy in the treatment of non-small cell lung cancer cell lines,particularly those with the EGFR mutation. The compounds of Formula Imay be advantageously co-administered with other anti-cancer agents inthe treatment of cancers characterized by over-expression of RAS, by anEGFR mutation, and/or by over-expression of AKT2. This co-administrationmay produce an improved treatment outcome as compared with soleadministration. This co-administration may even produce a synergisticbenefit, as previously defined, in treatment outcome as compared withsole administration.

COTI-2 and COTI-2MO5 are one and the same. COTI-2 and COTI-2MO5 are usedinterchangeably throughout the specification.

When introducing elements disclosed herein, the articles “a”, “an”,“the”, and “said” are intended to mean that there are one or more of theelements.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitation.

EXAMPLES

Synthesis of COTI-2

The synthesis of COTI-2, as depicted above, was conducted according tothe following synthetic methodology:

Imidazol-1-yl-(4-pyridin-2-yl-piperazin-1-yl)-methanethione (orintermediate 3 above) was formed as follows. N-(2-pyridyl)piperazine (MW163.22, 0.91 ml, 6.0 mmoles, 1 eq) 2 was added to a solution of1,1′-thiocarbonyldiimidazole (MW 178.22, 1.069 g, 6.0 mmoles, 1 eq) 1 in50 ml of dichloromethane at room temperature. The reaction mixture wasstirred overnight at room temperature. The mixture was washed withwater, dried over sodium sulfate, filtered and concentrated to provideimidazol-1-yl-(4-pyridin-2-yl-piperazin-1-yl)-methanethione (MW 273.36,1.354 g, 4.95 mmol, 83% yield) 3, which was used without furtherpurification. TLC(CH₂Cl₂/MeOH: 95/5): Rf=0.60, Product UV and Ninhydrinstain active. ¹H-NMR (400 MHz, CDCl₃), δ ppm: 3.72 (s, 4H), 4.02 (s,4H), 6.67 (d, 1H, J=7 Hz), 6.72 (dd, 1H, J=7 and 5 Hz), 7.11 (s, 1H),7.24 (s, 1H), 7.54 (t, 1H, J=7 Hz), 7.91 (s, 1H), 8.20 (d, 1H, J=5 Hz).

Hydrazine hydrate (MW 50.06, 0.26 ml, 5.44 mmoles, 1.1 eq) was added toa solution ofimidazol-1-yl-(4-pyridin-2-yl-piperazin-1-yl)-methanethione 3 (MW210.30, 1.040 g, 4.95 mmol, 1 eq) in 30 ml of ethanol at roomtemperature. The reaction mixture was stirred under reflux for 2 hours.A white precipitate formed. This white solid was filtered off and rinsedwith diethyl ether to yield 1-[N-(2-pyridyl)-piperazine)-carbothioicacid hydrazide (MW 237.33, 0.86 g, 3.62 mmol, 73% yield) 4 as a whitesolid, and used without further purification. TLC(CH₂Cl₂/MeOH: 95/5):Rf=0.20, Product UV and Ninhydrin stain active. ¹H-NMR (400 MHz,DMSO-d₆), δ? ppm: 3.53 (s, 4H), 3.85 (s, 4H), 6.66 (dd, 1H, J=8 and 5Hz), 6.82 (d, 1H, J=8 Hz), 7.55 (t, 1H, J=8 Hz), 8.12 (d, 1H, J=5 Hz).

Finally, COTI-2 was formed as follows.1-[N-(2-pyridyl)-piperazine)-carbothioic acid hydrazide (MW 237.33,0.475 g, 2.0 mmol, 1 eq) 4 and 6,7-dihydro-5H-quinolin-8-one (MW 147.18,0.306 g, 2.0 mmol, 1 eq) 5 was dissolved in 15 ml of ethanol at roomtemperature. The mixture was then stirred under reflux for 20 hours. Ayellow solid precipitated out of the solution. This solid was filteredoff then rinsed with methanol and diethyl ether to yield COTI-2 (MW366.48, 0.60 g, 1.64 mmol, 82% yield) as a yellow solid.TLC(CH₂Cl₂/MeOH: 95/5): Rf=0.75, Product UV and Ninhydrine stain active.HPLC analysis showed a mixture of isomers (approximately in 80/20ratio), and >98% purity. During the HPLC Method Development, asexpected, this product tends to be hydrolyzed in presence of TFA inmobile phase solution. MS (ESI+, 0.025% TFA in 50/50 MeOH/H₂O):[M+H]⁺=367.1, [M+Na]⁺=389.1; ¹H-NMR (400 MHz, CDCl₃), δ? ppm (Majorisomer): 2.09 (m, 2H), 2.92 (m, 4H), 3.67 (m, 4H), 4.27 (m, 4H), 6.69(dd, 1H, J=8 and 5 Hz), 7.25 (dd, 1H, J=8 and 5 Hz), 7.55 (d, 2H, J=8Hz), 8.23 (d, 1H, J=5 Hz), 8.63 (d, 1H, J=5 Hz), 14.76 (s, 1H). δ ppm(Minor isomer): 2.09 (m, 2H), 3.14 (t, 4H, J=6 Hz), 3.80 (m, 4H), 4.27(m, 4H), 6.66 (m, 1H), 7.31 (dd, 1H, J=8 and 5 Hz), 7.52 (m, 1H), 7.70(d, 1H, J=8 Hz), 8.23 (d, 1H, J=5 Hz), 8.53 (d, 1H, J=5 Hz), 15.65 (s,1H).

Synthesis of COTI-219

The synthesis of COTI-219, as depicted above, was conducted according tothe following synthetic methodology:

Imidazol-1-yl-(4-methyl-piperazin-1-yl)-methanethione (or intermediate 7above) was formed as follows. N-Methyl piperazine (MW 100.16, 0.67 ml,6.0 mmol, 1 eq) 6 was added to a solution of1,1′-thiocarbonyldiimidazole (MW 178.22, 1.069 g, 6.0 mmol, 1 eq) 1 in50 ml of dichloromethane at room temperature. The reaction mixture wasstirred overnight at room temperature. This mixture was washed withwater, dried over sodium sulfate, filtered and concentrated to provideimidazol-1-yl-(4-methyl-piperazin-1-yl)-methanethione (MW 210.30, 1.040g, 4.95 mmol, 82% yield) 7 and used without further purification.TLC(CH₂Cl₂/MeOH: 95/5): Rf=0.35, Product UV and Ninhydrine stain active.¹H-NMR (400 MHz, CDCl₃), δ ppm: 2.37 (s, 3H), 2.56 (s, 4H), 3.94 (s,4H), 7.11 (s, 1H), 7.21 (s, 1H), 7.88 (s, 1H).

1-(N-Methyl piperazine)-carbothioic acid hydrazide (or intermediate 8above) was formed as follows. Hydrazine hydrate (MW 50.06, 0.26 ml, 5.44mmol, 1.1 eq) was added to a solution ofimidazol-1-yl-(4-methyl-piperazin-1-yl)-methanethione 7 (MW 210.30,1.040 g, 4.95 mmol, 1 eq) in 30 ml of ethanol at room temperature. Thereaction mixture was stirred under reflux for 2 hours. This mixture wasconcentrated. The solid thus obtained was triturated with diethyl etherand filtered to yield 1-(N-Methyl piperazine)-carbothioic acid hydrazide(MW 174.27, 0.53 g, 3.04 mmol, 61% yield) 8 as a white solid which wasused without further purification. TLC(CH₂Cl₂/MeOH: 90/10): Rf=0.15,Product UV and Ninhydrin stain active. ¹H-NMR (400 MHz, DMSO-d₆), δ ppm:2.17 (s, 3H), 2.28 (t, 4H, J=5 Hz), 3.69 (t, 4H, J=5 Hz).

Finally, COTI-219 was made as follows. 1-(N-Methylpiperazine)-carbothioic acid hydrazide (MW 174.27, 0.174 g, 1.0 mmol, 1eq) 8 and 1,8-diazafluoren-9-one (MW 182.18, 0.182 g, 1.0 mmol, 1 eq) 9was dissolved in 15 ml of ethanol at room temperature, in the presenceof 1% glacial acetic acid (MW 60.05, 0.15 ml, 2.6 mmol, 2.6 eq). Themixture was stirred under reflux for 6 hours. After concentration, thecrude thus obtained was taken up in dichloromethane, washed with apotassium carbonate aqueous solution then with water. The organic layerwas dried over sodium sulfate, filtered and concentrated. The crude waspurified by ISCO CombiFlash™ Companion (Redisep™ cartridge 12 g, Normalphase, Gradient DCM/MeOH: 10/0 to 9/1) and provided COTI-219 (MW 338.43,0.330 g, 0.975 mmol, 98% yield) as a rust red solid. >95% purity by¹H-NMR. MS [ESI⁺, 90/10 MeOH/H₂O (5 mM NH₄OAc, 0.2% Acetic acid)]:[M+H]⁺=339.1, [M+Na]+=361.1; ¹H-NMR (400 MHz, CDCl₃), δ ppm: 2.31 (s,3H), 2.56 (t, 4H, J=5 Hz), 4.17 (t, 4H, J=5 Hz), 7.23 (dd, 1H, J=8 and 5Hz), 7.31 (dd, 1H, J=8 and 5 Hz), 7.86 (d, 1H, J=8 Hz), 7.97 (d, 1H, J=8Hz), 8.47 (d, 1H, J=5 Hz), 8.51 (d, 1H, J=5 Hz), 13.53 (s, 1H).

Synthesis of COTI-5

The synthesis of COTI-5, as depicted above, is conducted according tothe following synthetic methodology:

Intermediate 11 is formed by reacting compound 10 with potassium,permanganate under reflux conditions. Intermediate 11 is reacted withhydrazine hydrate in ethanol to form intermediate 12.

Intermediate 12 is reacted with Lawesson's reagent in dioxane to formintermediate 13.

Finally, COTI-5 is formed as follows. Intermediate 13 and6,7-dihydro-5H-quinolin-8-one 5 is dissolved in ethanol at roomtemperature to yield COTI-5.Synthesis of COTI-5The synthesis of COTI-5, as depicted above, is conducted according tothe following synthetic methodology:

Intermediate 14 is formed by irradiating compound 10 in the presence ofchlorine (the corresponding bromo compound of intermediate 14 can beformed using N-bromosuccinimide, benzyl peroxide in benzene).Intermediate 14 is reacted with S₈ and methyl iodide in TEA and DMF(PhSO₂Na, acetonitrile, Pr₄NBr at 80° C. for 24 h or S₈, t-BuOK at R.T.,THF then methyl iodide may also be used) to yield intermediate 15.Intermediate 15 is reacted with hydrazine hydrate in ethanol to formintermediate 13.

Finally, COTI-5 is formed as follows. Intermediate 13 and6,7-dihydro-5H-quinolin-8-one 5 is dissolved in ethanol at roomtemperature to yield COTI-5.

Example 1 In-Silico Assessment of Properties

An in-silico assessment of the properties of compounds according to thepresent invention was performed using the CHEMSAS® computationalplatform. CHEMSAS® is a robust proprietary computational platform foraccelerated drug discovery, optimization and lead selection based upon aunique combination of traditional and modern pharmacology principles,statistical modeling and machine learning technologies. At the centre ofthe CHEMSAS® platform is a hybrid machine learning technology that maybe used to: find, profile and optimize new targeted lead compounds; findnovel uses for known compounds; and, solve problems with existing orpotential drugs. In using the CHEMSAS® platform, first a therapeutictarget was selected, in this case cancer and more particularly SmallCell Lung Cancer. The second step involved the design of a candidatemolecule library containing thousands of potential compounds through theassembly of privileged molecular fragments. Thirdly, the candidatelibrary was profiled and optimized using a combination of validatedcomputational models and traditional expert medicinal chemistry. In thisstep, the CHEMSAS® platform developed 244 molecular descriptors for eachcandidate therapeutic compound. For example, molecular propertiesrelating to a candidate compound's therapeutic efficacy, expected humantoxicity, oral absorption, cumulative cellular resistance and/orkinetics were assessed. In some instances, comparative propertiesrelating to commercially relevant benchmark compounds were alsoassessed. Potential lead compounds were then selected from the candidatelibrary using a proprietary decision making tool designed to identifycandidates with the optimal physical chemical properties, efficacy,ADME/Toxicity profile, etc. according to a pre-determined set of designcriteria. The lead compounds selected from the candidate library werethen synthesized for further pre-clinical development.

The properties of certain compounds according to the present invention,specifically COTI-217, COTI-220, COTI-219, COTI-2 and COTI-5, that wereassessed in-silico using the CHEMSAS® computational platform are shownin Tables 1 to 13. Some of the predicted properties are validated by theexperimental data provided herein, while other properties have beenvalidated elsewhere during the development of other clinical candidates.The CHEMSAS® platform therefore provides a means of determining,predicting and/or testing the properties of a compound, particularlywhen used to determine the properties of compounds according to thepresent invention. The CHEMSAS® platform is also particularly useful incomparing the properties of compounds according to the invention withprior art compounds on a relative basis in silico.

Tables 1A and 1B: Physical Chemical Properties

Tables 1A and 1B shows that COTI-217, COTI-220, COTI-219, COTI-2 andCOTI-5 are “drug-like” with good drug like physical properties.

TABLE 1A MolID FORMULA MolWeight MnLogP HBndDon HBndAcc COTI217C17H22N6S 342.469 1.859199 1 6 COTI220 C18H20N6S 352.464 2.078432 1 6COTI219 C17H18N6S 338.437 1.7646 1 6 COTI2 C19H22N6S 366.491 3.041311 16 COTI5 C20H24N6S 380.518 2.22023 1 6

TABLE 1B MolID TPSA RotBnds LipinskiAlerts Veber COTI217 37.5435 3 0 0COTI220 53.3605 3 0 0 COTI219 53.3605 3 0 0 COTI2 53.3605 4 0 0 COTI553.3605 4 0 0 Legend forTable 1: Mol Weight stands for Molecular Weightmeasured in Daltons and is a size descriptor; MnLogP is an average ofMLogP, ALogP98 and CLogP, all of which are calculatedlipophilicity/solubility estimates; HBndDon stands for Hydrogen BondDonor and refers to the number of atoms able to donate electrons topotentially form Hydrogen bonds; HBndAcc stands for Hydrogen BondAcceptor and refers to the number of atoms able to accept electrons topotentially form Hydrogen bonds; TPSA stands for Topological PolarSurface Area and is a measure of Molecular Surface Charge/Polarity;RotBnds stands for Rotatable Bonds which is a count of freely rotatablesingle bonds in the molecule; Lipinski Alerts: If any 2 of (Molecularweight >500 Daltons, Hydrogen Bond Donors >5, Hydrogen BondAcceptors >10, MLogP >4.15) are true, then a molecule is likely to havepoor bioavailability; Veber Alerts: If TPSA >140 or number of RotatableBonds is >10, then bioavailability is likely to be poor.Tables 2A and 2B: Solubility Properties

Tables 2A and 2B shows that COTI-217, COTI-220, COTI-219, COTI-2 andCOTI-5 are expected to have acceptable solubility values for drug-likecompounds.

TABLE 2A MolID FORMULA MnLogP LogD (pH 7) LogS COTI217 C17H22N6S1.859199 0.309304 −3.09009 COTI220 C18H20N6S 2.078432 0.992417 −4.20136COTI219 C17H18N6S 1.7646 1.067558 −3.78407 COTI2 C19H22N6S 3.0413112.380243 −4.52904 COTI5 C20H24N6S 2.22023 1.019701 −4.49499

TABLE 2B MolID FORMULA Acid pKa 2 Base pKa 1 Base pKa 2 COTI217C17H22N6S None 7.65056 None COTI220 C18H20N6S None 7.65056 4.71559COTI219 C17H18N6S None 7.65056 3.90139 COTI2 C19H22N6S None 5.653564.882592 COTI5 C20H24N6S None 7.870707 5.617688 Legend for Table 2:MnLogP is an average of MLogP, ALogP98 and CLogP, all of which arecalculated lipophilicity/solubilty estimates; LogD (7.4) is a measure ofrelative solubility in octanol vs water at a specific pH, in this casepH = 7.4; LogS is the logarithm of the calculated solubility in purewater usually measured at 25 degrees centigrade; pKa is a calculatedestimate of the pH at which the drug or substructures of the drug is 50%ionized and 50% is unionized.

TABLE 3 Efficacy (LogGI50) Table 3 shows that COTI-217, COTI-220,COTI-219, COTI-2 and COTI-5 are predicted to have sub-micromolar invitro activity vs human SCLC cell lines. Actual measurements obtained invitro confirm the prediction of activity at sub-micromolar levels forCOTI-2 and COTI-219. MolID FORMULA DMS114 SHP-77 Predicted ActualCOTI217 C17H22N6S <-6 <-6 Active ND COTI220 C18H20N6S <-6 <-6 Active NDCOTI219 C17H18N6S <-6 <-6 Active Active COTI2 C19H22N6S <-6 <-6 ActiveActive COTI5 C20H24N6S <-6 <-6 Active ND Legend for Table 3: DMS114 is a“classical” human small cell lung cancer line that is maintained by theNational Cancer Institute in the United States; SHP-77 is a “variant”human small cell lung cancer line that is maintained by the NationalCancer Institute in the United States; Predicted is the predicted invitro Activity of the drug; Actual is the actual outcome of in vitrotesting in both of the reference human small cell lung cancer lines;“Active” refers to drugs with predicted or measured GI50 <1 μmol/L; NDmeans that the drug has not yet been tested in vitro.Tables 4A and 4B: Oral Absorption and BBB Penetration

Tables 4A and 4B shows that COTI-217, COTI-220, COTI-219, COTI-2 andCOTI-5 are expected to be absorbed orally.

TABLE 4A HIA- MolID FORMULA Mn % OrlAbs Min % Abs T2 (MD) COTI217C17H22N6S 82.67412 67.67412 2.16777 COTI220 C18H20N6S 88.79283 73.792830.144973 COTI219 C17H18N6S 85.52785 70.52785 0.314455 COTI2 C19H22N6S87.02755 72.02755 0.38029 COTI5 C20H24N6S 88.43881 73.43881 0.277855

TABLE 4B ProbBBB BBB- Clark SubKit MolID Pene LogBBB T2 (MD) LogBBBLogBB COTI217 0.918625 −0.32584 2.280528 −0.09599 −0.22923 COTI2200.26949 −0.24921 0.254967 −0.36111 −0.20053 COTI219 0.331 −0.390220.551314 −0.39876 −0.31048 COTI2 0.710661 −0.01576 0.416152 −0.19558−0.0185 COTI5 0.089884 −0.0646 0.315208 −0.37444 −0.05658 Legend forTable 4: Mn % OrlAbs is the prediction of the mean percent oralabsorption of the drug from an ensemble of 5-7 different models; Min %Abs is the minimum value for the Mn % OrlAbs at the lower 95% ConfidenceInterval; HIA-T2 (MD) is the Malanabois distance, which is a measure ofthe calculated statistical distance from the centre of a population ofdrugs with optimal oral absorption; ProbBBB Pene is an estimate of theprobability that the drug will penetrate the blood brain barrier andenter the central nervous system (CNS); BBB-T2 (MD) is the Malanaboisdistance, which is a measure of the calculated statistical distance fromthe centre of a population of drugs with optimal blood brain barrierpenetration; Clark LogBBB is an estimate of a drugs penetration of theblood brain barrier based on the drugs LogP and TPSA; SubKit LogBB isanother estimate of a drugs penetration of the blood brain barrier basedon the drugs LogP and TPSA; LogBB: if LogBB <= −1 the drug does notpentrate the BBB; if Log BB > 0 there is likely to be good BBpenetration; if −1 < logBB < 0 then BBB penetration is likely to bevariable and may be poor.

TABLE 5 Metabolic Stability (Per cent remaining at 60 minutes andcalculated half life in hours) Table 5 shows that in vitro metabolicstability is expected to be adequate for COTI-217, COTI-220, COTI-219,COTI-2 and COTI-5. COTI-2 is expected to be metabolized more quickly inhuman liver microsomes than the other COTI compounds. Both the estimatedT½ and the T½ measured in vitro for COTI-2 and 219 are good. Liver 95%CI In vitro MolID Microsomes Hepatocytes T½ hrs in Hrs T½ (Hrs) COTI21754 66.4 5.3 1.9-8.7 ND COTI220 64.1 72.5 3.9 1.4-6.4 ND COTI219 66.774.18 4 1.4-6.6 ~6.8(5.0, 7.0, 8.5) COTI2 23.7 55.94 8.7  3.1-14.3~6.0(1.7, 4.8, 11.4) COTI5 50.9 64.42 6.1 2.2-10  ND Legend for Table 5:Liver Microsomes is the estimated per cent remaining at 60 minutes afterintroduction of a dose of the drug into an in vitro/human livermicrosomal enzyme system; Hepatocytes is the estimated per centremaining at 60 minutes after introduction of a dose of the drug into anin vitro/human hepatocyte cellular system; T½ hrs is a calculatedestimate of the half life of the drug measured in hours; 95% CI in Hrsis the calculated 95% confidence interval estimate of the half life ofthe drug measured in hours; In vitro T½ (Hrs) is the actual half life inhours obtained from 3 in vitro experiments carried out at doses of 1μmol, 10 μmol and 100 μmol (in brackets).

TABLE 6 Probability (CYP450 isoenzyme Substrate) Table 6 shows thatCOTI-217, COTI-220, COTI-219, COTI-2 and COTI-5 are likely to bemetabolized by the CYP450 enzyme system. COTI- 217, COTI-220, COTI-219,COTI-2 and COTI-5 are expected to undergo at least some CYP3A457metabolism and COTI-2 may also undergo some CYP2D6 metabolism. MolIDFORMULA CYP1A2 CYP2B6 CYP2C8/9 CYP2C19 CYP2D6 CYP2E1 CYP3A457 COTI217C17H22N6S 0.57 0.03 0.08 0.05 0.84 0.03 0.51 COTI220 C18H20N6S 0.07 0.020.12 0.05 0.22 0.02 0.93 COTI219 C17H18N6S 0.34 0.03 0.15 0.06 0.52 0.030.6 COTI2 C19H22N6S 0.05 0.03 0.13 0.06 0.8 0.03 0.93 COTI5 C20H24N6S0.21 0.03 0.2 0.07 0.58 0.04 0.87 Legend for Table 6: Table 6 representsthe estimated probabilities that the drug in question will undergo atleast 20% of its phase 1 metabolism by one or more of the 7 majorisoenzyme forms of Cytochrome P450 (CYP450). The isoenzyme forms ofCYP450 in Table 6 are: 1A2, 2B6, 2C8 or 9, 2C19, 2D6, 2E1 and 3A4, 5 or7; these 7 isoenzyme forms account for >80% of phase 1 metabolism of alldrugs that are orally administered to humans. The majority of all orallyadministered drugs are metabolized by the CYP3A family of isoenzymes.

TABLE 7 Probability (CYP450 Iso enzyme Inhibitor) Table 7 shows thatCOTI-217, COTI-220, COTI-219, COTI-2 and COTI-5 are not expected tosignificantly inhibit any CYP450 isoenzyme. MolID FORMULA CYP1A2 CYP2B6CYP2C8/9 CYP2C19 CYP2D6 CYP2E1 CYP3A457 COTI217 C17H22N6S 0.1 0.06 0.080.07 0.22 0.07 0.22 COTI220 C18H20N6S 0.09 0.06 0.33 0.12 0.16 0.06 0.12COTI219 C17H18N6S 0.11 0.06 0.22 0.08 0.12 0.06 0.1 COTI2 C19H22N6S 0.090.06 0.33 0.18 0.37 0.07 0.4 COTI5 C20H24N6S 0.11 0.06 0.23 0.16 0.310.07 0.37 Legend for Table 7: Table 7 represents the estimatedprobabilities that the drug in question will inhibit a given CYPisoenzyme activity by at least 20%; the isoenzyme forms of CYP450 intable 7 are: 1A2, 2B6, 2C8 or 9, 2C19, 2D6, 2E1 and 3A4, 5 or 7; these 7isoenzyme forms account for >80% of phase 1 metabolism of all drugs thatare orally administered to humans.

TABLE 8 Probability (CYP450 Iso enzyme Inducer) Table 8 shows thatCOTI-217, COTI-220, COTI-219, COTI-2 and COTI-5 are not expected toinduce any of the CYP450 isoenzymes. MolID FORMULA CYP1A2 CYP2B6CYP2C8/9 CYP2C19 CYP2D6 CYP2E1 CYP3A457 COTI217 C17H22N6S 0.06 0.05 0.050.05 0.05 0.05 0.05 COTI220 C18H20N6S 0.23 0.05 0.05 0.05 0.05 0.05 0.05COTI219 C17H18N6S 0.06 0.05 0.05 0.05 0.05 0.05 0.07 COTI2 C19H22N6S0.07 0.05 0.05 0.05 0.05 0.05 0.09 COTI5 C20H24N6S 0.07 0.05 0.05 0.050.05 0.05 0.07 Legend for Table 8: Table 8 represents the estimatedprobabilities that the drug in question will induce a given CYPisoenzyme activity by at least 20%. The isoenzyme forms of CYP450 intable 8 are: 1A2, 2B6, 2C8 or 9, 2C19, 2D6, 2E1 and 3A4, 5 or 7; these 7isoenzyme forms account for >80% of phase 1 metabolism of all drugs thatare orally administered to humans.

TABLE 9 Probability of any Hepatic Toxicity Table 9 shows that COTI-217,COTI-220, COTI-219, COTI-2 and COTI-5 are not expected to cause HepaticToxicity. MolID FORMULA ProbHepTox1 ProbHepTox2 COTI217 C17H22N6S 0.1460.086 COTI220 C18H20N6S 0.082 0.47 COTI219 C17H18N6S 0.079 0.457 COTI2C19H22N6S 0.065 0.371 COTI5 C20H24N6S 0.099 0.252 Legend forTable 9:ProbHepTox1 is the average calculated probability from an ensemble ofmodels that the drug in question will cause liver toxicity; ProbHepTox2is the average calculated probability from a second, different ensembleof models that the drug in question will cause liver toxicity.

TABLE 10 Probability of P-glycoprotein Interaction Table 10 shows thatCOTI-217, COTI-220, COTI-219, COTI-2 and COTI-5 are expected to inhibitP-glycoprotein (P-gp) enzyme activity. COTI-2 and COTI-5 may also besubstrates for P-gp, whereas COTI-219 is relatively unlikely to be asubstrate for P-gp. P-gp is a protein expressed by many cancer cells andis felt to contribute to cellular resistance to many cancer drugs.Ideally, an effective cancer drug would either not be a substrate forP-gp or would inhibit P-gp activity, thereby reducing the likelihood ofP-gp related drug resistance. MolID FORMULA Substrate Inhibitor COTI217C17H22N6S 0.57 0.81 COTI220 C18H20N6S 0.62 0.87 COTI219 C17H18N6S 0.190.75 COTI2 C19H22N6S 0.79 0.9 COTI5 C20H24N6S 0.82 0.9 Legend for Table10: Table 10 represents the calculated probabilities from an ensemble ofmodels that the drug in question will interact with P-glycoprotein(P-gp) as a substrate or inhibitor.

TABLE 11 Animal and Human Toxicity Predictions Table 11 shows thatCOTI-217, COTI-220, COTI-219, COTI-2 and COTI-5 are expected to have lowto moderate acute toxicity as measured by LD50 when given by the oraland intraperitoneal route. Lower Lower ORL- IPR- IPR- MRTD MRTD MolIDFORMULA ORL-LD50 LD50 LD50 LD50 mg/kg/day mg/day COTI217 C17H22N6S 609.7192.8 139.6 44.2 2 120.5 COTI220 C18H20N6S 761.1 240.7 175.5 55.5 1.379.9 COTI219 C17H18N6S 1022 323.2 227.8 72 1.2 70.4 COTI2 C19H22N6S842.8 266.5 195.3 61.8 1.6 99 COTI5 C20H24N6S 773.9 244.7 151.5 47.9 1.167 Legend for Table 11: ORL-LD50 is the calculated point estimate of thedose of the drug in mg/kg that would cause death in 50% of healthy testlab rats when the drug is given orally; Lower ORL-LD50 is the calculatedlower 95% confidence interval point estimate of the dose of the drug inmg/kg that would cause death in 50% of healthy test lab rats when thedrug is given orally; IPR-LD50 is the calculated point estimate of thedose of the drug in mg/kg that would cause death in 50% of healthy testlab mice when the drug is given intraperitoneally; Lower ORL-LD50 is thecalculated lower 95% confidence interval point estimate of the dose ofthe drug in mg/kg that would cause death in 50% of healthy test lab micewhen the drug is given intraperitoneally; MRTD mg/kg/day is thecalculated maximum recommended therapeutic daily dose of the drug inmilligrams per kg per day for the average 60 Kg human adult; MRTD mg/dayis the calculated maximum recommended therapeutic daily dose of the drugin milligrams per day for the average 60 Kg human adult.

TABLE 12 Predicted hERG Interaction Table 12 shows that COTI-217,COTI-220, COTI-219, COTI-2 and COTI-5 are expected to have hERG IC50values of >1 μmol/l in keeping with a decreased risk of cardiactoxicity. In general, a hERG IC50 of <1 μmol/L would be associated withan increased probability of potential drug induced cardiac toxicity.IC50 MolID FORMULA (μmol) ProbIC50 > 1 μmol ProbIC50 > 10 COTI217C17H22N6S 2.6 0.88 0.06 COTI220 C18H20N6S 1.8 0.9 0.03 COTI219 C17H18N6S2.2 0.92 0.04 COTI2 C19H22N6S 1.6 0.92 0.02 COTI5 C20H24N6S 0.6 0.790.04 Legend for Table 12: IC50(μmol) is the calculated concentration ofthe drug that inhibits 50% of the activity of the hERG potassium channeland is an estimate of potential cardiac toxicity; ProbIC50 > 1 μmol isthe calculated probability that the IC50 for the drug with regards tothe hERG potassium channel is greater than 1 μmol/L; ProbIC50 > 10 μmolis the calculated probability that the IC50 for the drug with regards tothe hERG potassium channel is greater than 10 μmol/L;

TABLE 13 Predicted Genotoxicity Table 13 shows that COTI-2 and 219 areexpected to have a negative AMES test and that COTI-217, COTI-220,COTI-219, COTI-2 and COTI-5 are not expected to cause Polyploidicity inthe Guinea Pig cell model. MolID FORMULA ProbAMES+ PolyPldy COTI217C17H22N6S 0.94 0.15 COTI220 C18H20N6S 0.06 0.16 COTI219 C17H18N6S 0.060.15 COTI2 C19H22N6S 0.06 0.16 COTI5 C20H24N6S 0.06 0.23 Legend forTable 13: ProbAMES+ is the probability that the drug will induce arecognized gene mutation in a standard strain of cultured bacteria;PolyPldy is the probability that the drug will induce polyploidicity(i.e. an increased/abnormal number of chromosomes) in cultered guineapig cells.

Example 2 In Vitro Efficacy Against Various Cancer Cell Lines

To assess the efficacy of compounds according to the present inventionin the treatment of cancer, in vitro activity expressed as IC50(represents the concentration of an inhibitor that is required for 50%inhibition of its target, in nmol) was measured for several cancer celllines using standard methods for such tests known to persons skilled inthe art. Briefly, cells were plated in plastic tissue culture plates andgrown under standard conditions for each cell line, in carbondioxide/oxygen atmosphere in plastic tissue culture plates, in thepresence of COTI-2 or COTI-219 compounds at 35° C. for 3 days. Controlcultures were treated with vehicle minus compound. Cells were countedafter 3 days in culture and at a cell density of no more than 80%. Thefollowing cell lines, obtained from the National Cancer Institute, weretested: human SCLC cell lines DMS153, DMS114, SHP77; human NSCLC celllines H226, A460, A560; human breast cancer cell lines T47D, MCF7; humancolon cancer cell line HT29; and, human Leukemia cell lines K562, HL60.The results of these assays are presented in Table 14.

TABLE 14 in vitro IC50 against cancer cell lines COTI-2 IC50 COTI-219IC50 Cell Line Tumor Type (nM) (nM) SHP77 SCLC 156 +/− 8  787 +/− 61DMS153 SCLC 73 +/− 9 233 +/− 39 DMS114 SCLC 51 +/− 9 267 +/− 40 H226NSCLC 15000 +/− 1129 Not tested A460 NSCLC 7900 +/− 620 Not tested A549NSCLC 6300 +/− 671 Not tested T47D Breast Cancer 221 +/− 12 367 +/− 44MCF7 Breast Cancer 101 +/− 8  421 +/− 31 HT29 Colorectal Cancer 121 +/−11 403 +/− 32 K562 Leukemia 176 +/− 22 222 +/− 28 HL60 Leukemia 236 +/−9  374 +/− 46Table 14 shows that both COTI-2 and COTI-219 possess potent activity inthe low nanomolar range against SCLC tumor cell types, as well asseveral other tumor cell types such as breast cancer, colorectal cancerand Leukemia. Both drugs had an IC50 of less than 850 nM for the SHP77cell line, which is known to be resistant to several conventionaltherapeutic agents. COTI-2 did not possess nanomolar level activityagainst NSCLC cell types and COTI-219 was not tested against those celltypes. At least COTI-2 therefore exhibits selectivity in lung cancertreatment towards SCLC cell types. The in vitro data also confirms thein-silico predictions of efficacy, which estimated that less than 1 μM(1000 nM) would be required for efficacy in the SHP 77 and DMS 114 celllines.

Example 3 In Vivo Efficacy in SCLC Treatment

The nude mouse model of human SCLC was used to evaluate the in vivoefficacy of compounds of the present invention in comparison withseveral known chemotherapeutic agents. Nude mice were obtained form theNational Cancer Institute and the SHP-77 human SCLC cell line was chosenfor metastatic tumor xenografts. The control group consisted of 10animals, each of which were administered bilateral thigh injections of aprescribed volume of tumor cells. There were 6 treatment groups, eachcontaining 5 animals: COTI-2, COTI-4, COTI-219, Taxotere® (docetaxel),Cisplatin® (cis-diamminedichloroplatinum) and Tarceva® (erlotinib) Thetherapeutic agent was administered by intraperitoneal (IP) injection onalternate days beginning on Day 3 post tumor cell injection. Each animalin a treatment group was administered bilateral thigh injections withthe same prescribed volume of tumor cells as the control animals.Treatment continued for 31 days, following which the animals wereeuthanized and tissues were collected for subsequent analysis. The finaltumor size in mm³ is reported in FIG. 1 and the number of tumors isreported in FIG. 2.

Referring to FIG. 1, compounds according to the invention showed amarked decrease in tumor growth as compared with both the control andconventional agents. Control animals produced tumors having a meanvolume of 260+/−33 mm³. Animals treated with COTI-2 produced tumors ofmean volume 9.9 mm³, while those treated with COTI-219 produced tumorshaving mean volume 53+/−28 mm³. This compared well with those treatedwith Cisplatin®, which produced tumors having means volume 132+/−26 mm³and those treated with Taxotere®, which produced tumors having meanvolume 183 mm³. Animals treated with Tarceva® died before studyconclusion at 31 days.

Referring to FIG. 2, compounds according to the invention showed amarked decrease in number of tumors as compared with both the controland conventional agents. Control animals produced an average of 0.9tumors per injection site, whereas those treated with COTI-2 produced0.28, those treated with COTI-219 produced 0.38, those treated withCisplatin® produced 0.48 and those treated with Taxotere® produced 0.48.Animals treated with Tarceva® died before study conclusion at 31 days.

The above data show the efficacy of compounds according to the inventionin vivo against SCLC cell lines. Furthermore, compounds according to theinvention show better efficacy compared to conventionally administeredtherapeutic agents.

Example 4 In Vivo Effect of COTI-2 in SCLC Treatment on N417 TumorXenografts

Malignant N417 human SCLC cells in Matrigel™ were injectedsub-cutaneously into hind legs of nude mice and xenograft tumors wereallowed to grow to about 100 mm³. Mice were then administered dailyintraperitoneal injections with indicated concentrations of COTI-2 (inisotonic saline, as a cloudy liquid, total volume of 1 ml per injection)for one week. Tumor volumes were estimated by caliper measurement. Theresults are shown in FIG. 3.

Referring to FIG. 3, tumor volumes were graphed as means±standard error(SE).

A significant difference in tumor growth was observed at all dosagelevels. The decrease in efficacy seen at the 8 mg/kg level relative toother treatment levels is attributed to an error in solubilizing thecompound, since a small amount of undissolved material was observed atthe bottom of the treatment vial. Percentage values reported on FIG. 3are for efficacy of the compound expressed in terms of inhibition oftumor growth according to the following formula:(1−(Tf−Ti)/(Cf−Ci))*100wherein Tf is the final tumor volume, Ti is the initial tumor volume atthe onset of treatment, Cf is the final control tumor volume and Ci isthe initial control tumor volume at the onset of treatment. Even whenthe 8 mg/kg dose is included, tumor growth inhibition of 30% or more wasobserved across all dosage levels. It is noted that the N417 cell lineis generally regarded as the hardest SCLC cell line to treat. Thecompounds according to the invention therefore exhibit in vivo efficacyagainst a number of different SCLC cell lines.

Example 5 Resistance Testing

In order to evaluate the induction of resistance in vitro, compoundsaccording to the invention were tested in head to head comparisonsagainst conventional therapeutic agents Cisplatin® and Taxotere®. Thecompound designated COTI-4 (which is the subject of Applicant'sco-pending U.S. provisional patent application entitled “Composition andMethod for Treatment” filed Dec. 26, 2007) was also tested. Thestructure for COTI-4 is:

IC50 values were obtained using methods known to persons skilled in theart with two different human SCLC cell lines (DMS153 and SHP77) obtainedfrom the National Cancer Institute. The surviving 50% of cells from theinitial IC50 tested were harvested and cultured for 5 days, after whichtime this new generation of cells was re-treated with the same agent anda new IC50 value was established. The procedure was repeated for a totalof 5 generations. Emerging resistance was identified by increasing IC50values in successive generations. The results are shown in FIGS. 4 and 5(DMS153 and SHP77 cell lines, respectively), where the ordinate axis isprovided in terms of the ratio of the IC50 value to the IC50 value ofthe parental generation.

Referring to FIGS. 4 and 5, both COTI-2 and 219 exhibited little to noemerging resistance over 5 generations. This was in marked contrast tothe conventional therapies Cisplatin® and Taxotere® (labeled Paclitaxelin the figures), which showed significant increases in IC50 for bothcell lines. The SHP77 cell line in particular is known to be resistantto conventional agents; however, neither COTI 2 nor 219 showed anytendency towards resistance in this cell line. In fact, COTI-2demonstrated a statistically significant tendency to decrease resistance(IC50's less than 1 for successive generations) in both cell lines.COTI-2 therefore exhibits a collateral sensitivity whereby theresistance of cells is decreased over successive generations and thedrug might actually become more effective over time against these celllines. This corroborates the in-silico predictions for COTI-2 and 219;COTI-2 was predicted to be a strong P-glycoprotein inhibitor, which isconsistent with decreasing the tendency towards drug resistance, whereasCOTI-219 was predicted to be both a P-glycoprotein inhibitor and/or aweak substrate for P-glycoprotein, also consistent with minimalaccumulation in resistance over successive generations. The in-silicopredictions for resistance profile of compounds according to theinvention are therefore confirmed by these assays.

Example 6 In Vitro Efficacy in Brain Cancer

In order to determine the efficacy of the present invention againsthuman Glioma and Astrocytoma cell lines, IC50 values were determined byin vitro assay of four malignant human brain cancer cell lines (U87MG,grade III glioblastoma/astrocytoma; SNB-19, glioma/astrocytoma Grade IV,glioblastoma multiforme; SF-268, glioma; SF-295, glioma). Human braincancers are notoriously difficult to treat.

Cell lines were obtained from the Human Tissue Culture Collection(ATCC), grown and maintained under ATCC-specified conditions, and testedto ensure viability and lack of contaminating mycoplasma and commonviruses. Healthy cells were plated in 96-well culture plates in mediumplus fetal bovine serum and allowed to adhere for 16 h, followed byaddition of COTI-2, COTI-219, Cisplatin®, or BCNU(1,3-Bis(2-chloroethyl)-1-nitrosourea) at multiple concentrationsranging from those that had no effect on proliferation to those thatinhibited proliferation by 90% or more. A viability stain (alamar Blue)was added to cells after 4-7 days of drug exposure (approximately 4doublings of control cells; maximum cell density in wells approximately80%), and assayed for total cellular metabolic activity (a function ofpopulation density of live cells) by absorbance. Concentrations of theagent required to inhibit proliferation by 50% (IC50 value) were derivedby interpolation of plotted data (mean values derived from 3 independentexperiments standard error). Results are reported in Table 15.

TABLE 15 IC50 values for Human Glioma/Astrocytoma cell Lines COTI-2COTI-219 Cisplatin BCNU Cell Line (nM) (nM) (nM) (nM) U87 48 +/− 92370+/− 490 +/− 9  1520 +/− 130 SNB-19  8 +/− 3 1990+/− 870 +/− 40 2250+/− 700 SF-268 66 +/− 8 1170+/− Not tested Not tested SF-295 184 +/− 232390+/− Not tested Not tested

At least the COTI-2 compounds were shown to have better efficacy againstglioma/astrocytoma cell lines as compared with the conventional agentsCisplatin® and BCNU. COTI-2 showed an order of magnitude greaterefficacy than Cisplatin® against U87 and two orders of magnitude greaterefficacy against SNB-19. These results show that at least COTI-2compounds have efficacy against glioma/astrocytoma cell lines.

Example 7 In Vivo Effect of COTI-2 on Cancerous Brain Tumours

Malignant U87 human glioma (brain tumour) cells in Matrigel™ wereinjected sub-cutaneously into hind legs of nude mice, allowed to grow to200-300 mm³, then treated 3 times per week (Mon, Wed, Fri) withindicated concentrations of COTI-2 (in isotonic saline, as a cloudyliquid, total volume of 1 ml per injection). Tumour volumes wereestimated by caliper measurement. The results are shown in FIGS. 6A and6B.

In FIG. 6A, tumour volumes were graphed as means±standard error (SE)(n=11-14 for each data point). The asterisk indicates a significantdifference (p<0.05) between the 8 mg/kg treatment group and both thesaline control and 4 mg/kg treatment groups. There was no significantdifference between the 4 mg/kg group and the saline control group.

In FIG. 6B, tumour volumes were graphed as fractional increase involume, to correct for differences in starting volume, ±SE. The asteriskindicates a significant difference (p<0.05) between the 8 mg/kgtreatment group and both the saline control and 4 mg/kg treatmentgroups. There was no significant difference between the 4 mg/kg groupand the saline control group. The flag (

) indicates a significant difference between the 8 mg/kg group and thesaline group, but not between the 8 mg/kg group and the 4 mg/kg group.

FIGS. 6A and 6B show that compounds of the present invention areeffective in the treatment of established human brain tumors. Thecompounds delayed tumor growth by about 25% at a dosage of 8 mg/kg givenjust three times per week. Although no significant effect was observedat a dosage of 4 mg/kg, more frequent administration may have produced asignificant effect at this dosage.

Example 8 Toxicity Testing

An escalating dose acute toxicity study was conducted with COTI-2,COTI-4 and COTI-219. Standard lab mice were divided into four treatmentgroups (control, 4, 8, 16 mg/kg) with four animals per group. It shouldbe noted that the highest dose was approximately 10 times the estimatedeffective dose. Mice were given alternate day IP injections for 28 days.Weight loss/gain of the mice was measured and the mice were observed foradverse effects such as vomiting, diarrhea, seizures, etc. Blood andtissue samples were harvested for histopathology. None of the drugsproduced any weight loss at any of the administered doses over theentire 28 day period. No evidence of acute toxicity was obtained and noadverse effects were observed. The compounds according to the presentinvention are therefore believed to be safe and non-toxic.

Example 9 In Vitro Metabolic Stability in Human Liver Microsomes

To evaluate the stability of these compounds in terms of clearance bythe liver, human liver microsomes (HLM) at a concentration of 0.5 mg/mlwere incubated with 0.823 mM NADPH, 5 mM UDPGA, 1 mM MgCl₂ and COTI-2 or219 at concentrations of 1, 10 and 100 μM. Sampling was conducted at 1,20, 40, 60, 120,180 and 240 minutes and the remaining concentration ofeach compound was evaluated. A half life (T_(1/2)) was calculated ateach concentration, along with the rate of clearance by the liver(C_(L)). The results are provided in Table 16 for each compound. TheC_(L) values compare favorably with published values for other marketedtherapeutic agents under identical conditions. The half life ofcompounds according to the invention is therefore likely to be longenough to permit convenient dosing, while not being so long as to leadto accumulation in patients with potential long term toxicity effects.

TABLE 16 Half-life and Liver Clearance Rate by HLM at 0.5 mg/mLConcentration C_(L) Compound (μM) T_(1/2) (min) (μL/min/mg) COTI-2 1102.1 12 10 285.7 4 100 683.1 2 COTI-219 1 301.2 4.2 10 420.7 3 100508.5 2.5

The average half life for COTI-2 was 6 hours and for COTI-219 was 6.8hours. The in-silico prediction for CL in the 95% confidence intervalwas from 3.1-14.3 for COTI-2 and from 1.4-6.6 for COTI-219; thiscompares well with the data presented in Table 3.

Example 10 Mechanism of Action

Without wishing to be limited by theory, it is believed that moleculesaccording to the present invention, particularly COTI-2, act in thetreatment of cancer in a manner consistent with the followingmechanistic observations. The following observations were obtained usinggene expression profiling techniques and targeted in vitro testing.Molecules of the present invention are believed to function as kinaseinhibitors. Molecules of the present invention are also believed tofunction as promoters of apoptosis. Promotion of apoptosis is achievedby decreasing phosphorylation of Caspase 9; this has the effect ofincreasing active Caspase 9 and inducing apoptosis via Caspase 3.

To confirm this mechanism SHP77 cells were treated with 250 nM of COTI-2and incubated for 3 and 6 hours. Western blots of the cellular lysatesare presented in FIG. 7. Phospho-Akt expression was decreased ascompared to control at both 3 and 6 hours, with corresponding increasesin Akt levels. There was no change in phospho-STAT3 expression, althougha slight decrease in total STAT3 (˜30%) was observed at 6 hrs. There wasno observed reactivation of Caspase 8; its level of expression remainedconstant in treated and control cells. However, the most dramatic changewas a profound suppression of phospho-Caspase 9 at both 3 and 6 hrs ofincubation. These results confirm the proposed mechanism of action.

Example 11 In-silico Comparative Data

The in-silico model was used to test properties of compounds describedin PCT Publication No. WO2006/009765: NSC716768, NSC73306, NSC73303,NSC668496, and NSC693323. Compounds JBC271A, JBC271B (Journal ofBiological Chemistry 271, 13515-13522 (1996)) and JICS75 (Journal of theIndian Chemical Society, 75, 392-394 (1998) and Journal of the IndianChemical Society, 72, 403-405 (1995)) are as follows:

Results of in-silico testing are shown in Tables 17 to 20. The legendsfor these tables correspond to those of Example 1, except whereindicated, and the methodology used to create the Tables was identical.Tables 17A and 17B: Physical Chemical PropertiesTable 17 shows that all tested compounds are drug like with no alertsfor poor absorption or bioavailability.

TABLE 17A MolID FORMULA MolWeight MnLogP HBnd Don HBnd Acc NSC716768C17H20N6O4S 404.449 2.082079 2 10 NSC73306 C16H12Cl2N4O2S 395.2683.155598 3 6 NSC73303 C15H12N4OS 296.352 2.564086 3 5 NSC668496C15H18N4OS 302.4 2.541123 2 5 NSC693323 C14H24N6S2 340.516 2.39891 2 6JBC271A C8H12N4O2S2 260.338 0.257966 2 6 JBC271B C9H14N4O2S2 274.3650.542592 1 6 JICS75 C11H19N3OS 241.357 1.600519 1 4

TABLE 17B MolID FORMULA TPSA RotBnds Lipinski Alerts Veber NSC716768C17H20N6O4S 112.7027 7 0 0 NSC73306 C16H12Cl2N4O2S 75.9848 5 0 0NSC73303 C15H12N4OS 67.0547 4 0 0 NSC668496 C15H18N4OS 57.597 3 0 0NSC693323 C14H24N6S2 54.972 7 0 0 JBC271A C8H12N4O2S2 66.5271 3 0 0JBC271B C9H14N4O2S2 57.0694 3 0 0 JICS75 C11H19N3OS 36.4161 3 0 0

TABLE 18 Solubility Properties Table 18 shows that all tested compoundshave acceptable and comparable solubility with the COTI compounds exceptfor NSC73306 which would be expected to have very poor water solubility.MolID FORMULA MnLogP LogS NSC716768 C17H20N6O4S 2.082079 −3.46551NSC73306 C16H12Cl2N4O2S 3.155598 −5.76993 NSC73303 C15H12N4OS 2.564086−3.7869 NSC668496 C15H18N4OS 2.541123 −3.87371 NSC693323 C14H24N6S22.39891 −3.27041 JBC271A C8H12N4O2S2 0.257966 −1.76143 JBC271BC9H14N4O2S2 0.542592 −1.83773 JICS75 C11H19N3OS 1.600519 −2.45438

TABLE 19 Efficacy (LogGI50) Table 19 shows that all tested compoundsexcept for NSC693323 are predicted to be inactive against human SCLCcell lines DMS114 and SHP-77 in vitro. Therefore, there is no rationalefor use of any of the tested compounds except for NSC693323 astherapeutic agents in the treatment of SCLC. NSC693323 has an averageGI50 of −6.3. By comparison, COTI-2 has LOG(GI50) for DMS114 determinedin vitro of −7.2 to −7.4, representing ~10 times better in vitroefficacy than the predictions for NSC693323 Mean Over NCI/DTP DMS SHP-60 cell MolID FORMULA 114 77 Predicted line panel NSC716768 C17H20N6O4S<-6 <-6 Inactive −4.7 NSC73306 C16H12Cl2N4O2S <-6 <-6 Inactive −4.9NSC73303 C15H12N4OS <-6 <-6 Inactive ND NSC668496 C15H18N4OS <-6 <-6Inactive −6.1 NSC693323 C14H24N6S2 <-6 <-6 Active −6.3 JBC271AC8H12N4O2S2 <-6 <-6 Inactive ND JBC271B C9H14N4O2S2 <-6 <-6 Inactive NDJICS75 C11H19N3OS <-6 <-6 Inactive ND Legend for Table 19: Mean OverNCI/DTP 60 cell line panel is the mean of the GI50's for all 60 celllines NOT including DMS114 and SHP-77; ND means not done/not available.Table 20: Oral Absorption and BBB Penetration

Table 20 shows that all tested compounds are predicted to have good oralabsorption with variable to poor CNS penetration. The only potentiallyactive drug, NSC693323, likely penetrates into the CNS poorly.

TABLE 20 MolID FORMULA Mn % OrlAbs Min % Abs HIA-T2(MD) NSC716768C17H20N6O4S 86.33807 71.33807 3.556507 NSC73306 C16H12Cl2N4O2S 73.4351258.43512 2.075257 NSC73303 C15H12N4OS 88.14632 73.14632 0.078544NSC668496 C15H18N4OS 87.81207 72.81207 0.055115 NSC693323 C14H24N6S284.59752 69.59752 0.097439 JBC271A C8H12N4O2S2 80.28443 65.284432.273772 JBC271B C9H14N4O2S2 84.04259 69.04259 2.267253 JICS75C11H19N3OS 91.74003 76.74003 2.023605

TABLE 20B BBB- MolID FORMULA ProbBBBPene LogBBB T2(MD) NSC716768C17H20N6O4S 0.009519 <<−1.00 9.681481 NSC73306 C16H12C12N4O2S 0.051291−0.1554 4.758413 NSC73303 C15H12N4OS 0.359669 −0.41974 1.216003NSC668496 C15H18N4OS 0.306419 −0.26927 0.426904 NSC693323 C14H24N6S20.265543 −0.24742 0.294411 JBC271A C8H12N4O2S2 0.818135 −1.124833.888207 JBC271B C9H14N4O2S2 0.806343 −0.91155 3.439832 JICS75C11H19N3OS 0.840636 −0.25614 1.981566

Example 12 AKT Kinase Activity Inhibition

Methods

Cell Lines

DMS-114 and SHP-77 SCLC cell lines were routinely grown in RPMI mediasupplemented with 10% fetal calf serum and antibiotics to preventbacterial growth, and maintained by twice-weekly passaging at a ratio of1:3. Typically, 0.5−1.0×10⁶ cells were incubated with COTI-2 or anequivalent volume (2-5 μL) of DMSO to observe changes in Akt activity orgene expression.

Measurement of Akt Kinase Activity

Cells were incubated in 10 cm dishes with DMSO or 250 nM of COTI-2 for2, 4, 8, 16, or 24 hrs after which kinase activity was measured with anon-radioactive Akt kinase kit (cat #9840) from Cell Signaling. Briefly,after lysing the cells in a protein non-denaturing buffer, Akt isimmunoprecipitated with immobilized antibody. The kinase reactioncatalyzed by immunoprecipitated Akt consists of in vitro phosphorylationof supplemented recombinant GSK-3, a natural in vivo Akt substrate, inthe presence of added ATP. The amount of phosphorylated GSK-3 isdetermined by western blot analysis with phospho-GSK-3αβ(Ser21/9)antibody.

Measurement of Total Akt by Western Blot Analysis

Duplicate 10 cm tissue culture dishes seeded with 0.5−1.0×10⁶ cells wereincubated with 250 nM of COTI-2 for 0, 2, 4, 8, 16, 24, 48, and 96 hrs.Cell viability was determined at 24, 48, and 96 hrs by the trypanexclusion method. Whole cell lysates were prepared using standardprocedures and fifty micrograms of lysate protein was separated onSDS-PAGE gels and analyzed by western blotting.

Results and Discussion

COTI-2 is an inhibitor of Akt kinase activity in SCLC cell lines

Incubation of DMS-114 and SHP-77 cells with 250 nM of COTI-2 resulted ina time-related decrease in Akt kinase activity starting at 2 hrs andextending over the 24 hrs duration of the experiment. In DMS-114 cellsthe kinase activity was below the threshold for measurement at 8 hrs andremained significantly lower relative to the control group at 24 hrs(FIG. 8). In SHP-77 cells there was a more gradual decrease in kinaseactivity over the course of the experiment, with a tendency for theactivity to return towards control levels after 24 hrs of incubation(FIG. 8). In both cell lines, total Akt protein decreased after 24 hrsof incubation with COTI-2, analogous to the changes in kinase activityobserved at this time (FIG. 9). Cell viability assays using trypan blueexclusion confirmed that this dose of COTI-2 effectively kills up to 80%of the original tumor cells within four days of incubation. Theseresults indicate that COTI-2 inhibits Akt kinase activity either bypreventing activation of Akt (directly or indirectly) or by preventingAkt from activating its downstream target(s).

Example 13 mTOR-Rictor Complex Formation

Mechanism of COTI-2 Apoptosis in SCLC Cells: Evidence from KinaseExperiments and Transcriptome Analysis

In order to confirm the involvement of AKT in COTI-2 induced apoptosis,proteomic analyses of time-dependent changes in AKT kinase activity andAKT phosphorylation was performed using 2 SCLC cell lines (DMS114 andSHP77). Furthermore, to identify additional mechanistic targets for thiscompound, microarray experiments were followed by differential geneexpression analysis. In summary, the results showed that, in response toCOTI-2, AKT is down-regulated at the gene expression, whole protein, andkinase levels, thus showing that this critical cellular regulator is adirect/indirect target for COTI-2. Furthermore, the down-regulation ofmTOR-Rictor, proposed to phosphorylate AKT at Ser473 site, by COTI-2provides additional evidence for the proposed mechanism of action ofthis compound.

Methods:

Cell Lines:

DMS114 and SHP77 cell lines were routinely grown in RPMI mediasupplemented with 10% fetal calf serum and antibiotics to preventbacterial growth, and maintained by twice-weekly passaging at a ratio of1:3. Typically, 0.5−1.0×10⁶ cells were incubated with COTI-2 or anequivalent volume (2-5 μl) of DMSO to observe changes in AKT activity orgene expression.

Measurement of AKT Kinase Activity:

Cells were incubated in 10 cm dishes with DMSO or 250 nM of COTI-2 for2, 4, 8, 16, or 24 h after which kinase activity was measured with anon-radioactive AKT kinase kit (cat #9840) from Cell Signaling. Briefly,after lysing the cells in a protein non-denaturing buffer, AKT wasimmunoprecipitated with immobilized antibody. The kinase reactioncatalyzed by immunoprecipitated AKT consists of in vitro phosphorylationof supplemented recombinant GSK-3, a natural in vivo AKT substrate, inthe presence of added ATP. The amount of phosphorylated GSK-3 wasdetermined by western blot analysis with phospho-GSK-3αβ(Ser21/9)antibody.

Measurement of Total Akt by Western Blot Analysis:

Duplicate 10 cm tissue culture dishes seeded with 0.5−1.0×10⁶ cells wereincubated with 250 nM of COTI-2 for 0, 2, 4, 8, 16, 48, and 96 h. Cellviability was determined at 24, 48, and 96 h by the trypan exclusionmethod. Whole cell lysates were prepared using standard procedures and50 μg of lysate protein was separated on SDS-PAGE gels and analyzed bywestern blotting.

Microarray Experiments:

For each cell line, four sets of microarray experiments were performed,with each set consisting of control and treated cells exposed for 6 heither to DMSO or COTI-2 at 150 or 300 nM total concentration. Total RNAwas prepared with RNeasy (Qiagen) and converted to Cy3 or Cy5-labelledcRNA according to procedures provided with the linear amplification kitfrom Agilent Technologies. Two hundred micrograms of labeled cRNA werehybridized to 4×44K Agilent whole genome microarrays, the arrays werewashed, scanned and relevant features were extracted according to themanufacturer's protocol and further analyzed as described by Wolber etal. (Methods in Enzymology 410:28-57), which is incorporated herein byreference in its entirety.

Gene Expression Analysis:

Cy5 and Cy3 features were extracted and normalized by median centeringthe expression values relative to the median value of the 8hybridization experiments performed on each microarray slide. Thus, foreach gene, 4 control and 4 treatment values (two each for 150 and 300nM) were obtained. Quality control (QC) reports showed that data was ofgood quality and spot saturation or abnormal features were seldomobserved. Statistically significant changes in gene expression betweentreatment and control groups were determined by SAM analysis using TIGRsoftware from The Institute of Genomic Research or by t tests with falsediscovery correction. Only genes that were differentially expressed inall 4 biologically-independent groups were further analyzed. (Anindependent analysis of gene expression at the lower 150 nM level ofCOTI-2 yield additional results reflecting changes in gene expression atthis lower concentration). Gene ontology analysis of the differentiallyexpressed genes was performed as previously described (Bioinformatics 8:426). Statistical analysis of ontology-based gene overrepresentationbetween DMS114 and SHP77 cell lines was performed as described by AlSharour et al. (Nucleic Acids Res. 35: W91-W96). The intersection ofcommon genes was determined using GeneVenn(http://mcbc.usm.edu/qenevenn/genevenn.htm). Network analysis wasperformed as described by Rhodes et al. (Natl Biotechnol. 23: 951-959)and Franke et al. (Am J Hum Genet. 78: 1011-1025) Overrepresentation oftranscription factor binding sites in the promoters of up- ordown-regulated genes was calculated as described by Ho Sui et al.(Nucleic Acids Res. 33: 3154-3164).

Results and Discussion:

The AKT kinase activity was determined by measuring the phosphorylationof GSK-3, which is a downstream target of activated AKT. Incubation ofDMS114 and SHP77 cells with 250 nM of COTI-2 resulted in a time-relateddecrease in AKT activity starting at 2 h and extending over the 24-hduration of the experiment (FIG. 8). In DMS114 cells, the kinaseactivity was below the threshold for measurement at 8 h and remainedsignificantly lower relative to the control group at 24 h. In SHP77cells, there was a more gradual decrease in kinase activity over thecourse of the experiment, with a tendency for the activity to returntowards control levels after 24 h of incubation. In both cell lines,total AKT protein decreased after 24 h of incubation with COTI-2,analogous to the changes in kinase activity observed at this time (FIG.9).

The transcriptional activity of DMS114 and SHP77 cell lines weredocumented following a 6-h incubation with COTI-2 (150 or 300 nM) orvehicle only control. A number of genes were differentially regulated inboth cell lines (FIG. 10). Of particular interest, the AKT geneexpression data (FIGS. 11A-11B) is consistent with the observeddown-regulation of AKT protein observed by western blot analysis (FIG.9), and the down-regulation of Rictor (FIG. 11C) is also consistent withthe observed decreased AKT kinase activity (FIG. 8). Interestingly, theanalysis of the promoters of the differentially regulated genesindicates an over-representation of promoters regulated by the forkheadfamily of transcriptional factors. These data provide additionalevidence for AKT as the target of COTI-2, since AKT inactivates thesetranscriptional factors via phosphorylation; therefore, the inactivationof AKT by COTI-2 would expectedly lead to the dysregulation of genesregulated by the forkhead transcription factors.

Example 14 COTI-2 Induced Apoptosis does not Involve Downregulated PI3KActivity but is associated with decreased mTOR Activation

This determined whether COTI-2 interfered with the upstream regulatorsof AKT, namely PI3 kinase 1, PDK1, and mTOR-Rictor complexes (“PDK2”activity). Four cancer cell lines, A549 & H460 (NSCLC) and DMS114 &SHP77 (SCLC) were utilized.

Methods:

Cells were grown to 60-80% confluency in RPMI media supplemented with10% fetal calf serum and antibiotics to prevent bacterial growth. 1×10⁶cells were incubated with 100, 250, 500, or 1000 nM COTI-2 either for 4h, to observe the effect on kinase-mediated events, or for 24 h, toobserve the effect on the levels of pro/anti-apoptotic molecules. As anegative control, an equivalent volume of DMSO was utilized. Whole celllysates were prepared using standard procedures and 50 μg of lysateprotein was separated on SDS-PAGE gels and analyzed by western blotting.To measure PI3-K activity, a commercial kit was purchased from Echelon.Following the procedures recommended by the manufacturer, membranelipids were extracted in several steps and the resulting PIP3 wasimmobilized to nitrocellulose and measured by its interaction with aPH-domain containing protein and HRP-conjugated antibody supplied withthe kit. As an additional control in these experiments, the specificPI3-K inhibitor, LY294002 was used to inhibit PI3-K and thus stop PIP3production.

To determine the effect of 250 nM concentration of COTI-2 on PI3-Kactivity, cells grown to 60-80% confluence in 10 cm cell culture disheswere treated with COTI-2 or an equivalent volume of DMSO. In addition,controls were pre-incubated with 50 μM of the PI3-K inhibitor LY294002for 15 minutes prior to the addition of COTI-2. Treatment with COTI-2lasted for 20 minutes after which the cells were processed for PIP3detection according to Echelon's protocol.

Results and Discussion:

COTI-2 has Minimal Effect on PI3-K Activity in SCLC Cells:

To determine the effect of COTI-2 on PI3K activity, both DMS114 andSHP77 cells were grown and exposed to 250 nM COTI-2 alone, 250 nMCOTI-2+LY294002, or DMSO (solvent) alone. Subsequently, the PIP3 levelswere detected. The PIP3 levels are an indication of the activity of PI3Ksince PI3K converts inactive PIP2 to active PIP3, which is then able tobring Akt and its activators (PDK1 and PDK2) into close proximity in thecell membrane. As expected, the results indicated that in the presenceof the PI3K-inhibitor, LY204002, there was little detectable PIP3 inDMS114 cells (FIG. 12). However, LY204002 did not inhibit PI3K activityin SHP77 cells since approximately 14 pmol of PIP3 was detected. Thisdata can be explained by the non-specificity of LY204002 for PI3K, sincethis inhibitor was shown to inhibit PI3K-related kinases, for examplemTOR (EMBO J. 15: 5256-5267 and Cancer Research 59: 2581-2586).Alternatively, it is also possible that PI3-K is mutated in this cellline and thus unable to respond to the inhibitor as well as to COTI-2.Several mutations that abolish PI3-K activity have been described andare catalogued in the human protein mutation database MutDB(http://mutdb.org/). Nevertheless, COTI-2 alone did not inhibit PI3Kactivity as indicated by the PIP3 levels in both DMS114 and SHP77 celllines (FIG. 12). In fact, there was a small increase in PI3K activity inDMS114 cells as evidenced by approximately 25% increase in activity.This data is also consistent with the hypothesis that AKT is the director indirect target of COTI-2 because inhibiting AKT results in theinhibition of its downstream targets mTORC1 and S6K (S6 kinase 1), whichare involved in a negative-feedback loop that blocks signaling to PI3K(Biochem. Soc. Trans. 37: 217-222). Therefore, the inhibition of AKTprevents the activation of the negative-feedback loop and allows theincreased activity of PI3K.

In conclusion, since COTI-2 does not act on PI3K, it does not inhibitAKT via upstream regulation. COTI-2 therefore seems to prevent theactivation of AKT through direct interaction.

COTI-2 Impairs mTOR Activation and Decreases Phospho-Thr308 Levels ofPKB/Akt:

Four cancer cell lines, namely A549 & H460 (NSCLC) and DMS114 & SHP77(SCLC), were treated for 4 h with various concentrations of COTI-2 orDMSO (solvent). Similar levels of total mTOR, before and afterincubation with COTI-2, were observed in A549, H460, and DMS114 celllines (FIG. 13). In SHP77 cells, more than in the other cell lines, ahigher order complex that reacted positive with mTOR antibody, but notwith other antibodies, accumulated at the bottom of the loading wellsand was not resolved by SDS-PAGE. Presumably because of this, less mTORwas detected at the appropriate molecular weight on the western blot.One possible interpretation of this finding is that in this cell line,COTI-2 could preferentially bind to mTOR or to one of its complexes withRictor, Raptor, or other proteins, resulting in large protein aggregatesthat may be functionally impaired. Consistent with this, the level ofmTOR phospho-Ser2448 that is necessary for mTOR activity was alsoreduced in this cell line. Taken together with previous results showingthat COTI-2 decreases phospho-Ser473 levels of PKB/Akt, these findingssuggested that in SCLC cells, COTI-2 inhibits mTOR activity, andconsequently the activity of PKB/Akt. PKB/Akt phospho-Thr308 levelsdecreased in A549 cells (and to a lesser extent in SHP 77 cells, shownby the asterisk) after incubation with COTI-2.

These experiments provide further evidence that COTI-2 acts to inhibitAkt phosphorylation through mTOR-Rictor complex formation.

Example 15 Synergistic Effect with mTOR Inhibitors

Effects of Temsirolimus and Rapamycin on the Cytotoxicity of COTI-219and COTI-2 Against the Human Glioblastoma Cell Line U87

In vitro IC50 values were obtained for combinations of compounds of thepresent invention with temsirolimus or rapamycin in the treatment of U87glioblastoma cells. Temsirolimus and rapamycin both inhibitproliferation of human U87 glioblastoma cells, as single agents (FIG.14). FIG. 14 shows the typical dose-response of U87 cells totemsirolimus or rapamycin. Both inhibit proliferation of these cellswith increasing concentration. Over triplicate experiments: IC₅₀(Temsirolimus)=3.6 μM±1.4 μM and IC₅₀ (Rapamycin)=9.9 μM±1.8 μM.

It was found that both temsirolimus and rapamycin, in combination withCOTI-219, exert greater-than-additive growth inhibitory effects on U87cells. Both temsirolimus and rapamycin, in combination with COTI-2 (atCOTI-2 concentrations less than the IC50), exert somegreater-than-additive growth inhibitory effects on U87 cells.

In Combination with COTI-219, Temsirolimus Had a Greater-than-AdditiveInhibitory Effect on Proliferation of U87 Glioblastoma Cells

Temsirolimus was provided in liquid form from the London Regional CancerProgram (LRCP) Pharmacy. COTI-219 was prepared as a 50 mM stock in DMSO,then diluted to 10 mM in DMSO followed by serial dilution water foraddition to medium overlaying cells. Cells were incubated withtemsirolimus in combination with COTI-219 for 4 days (in wells of 96well plates) followed by analysis of live cell density using thefluorescent vital stain alamarBlue.

Referring to FIG. 15, relative cell density was plotted againstconcentration of COTI-219, normalized to the inhibitory effect oftemsirolimus alone (this ranged from 20-30% inhibition at very lowconcentrations to 80-90% inhibition at the higher end of concentrationsused in this study). Thus, data points indicating proliferation lessthan control values indicate greater-than-additive inhibition ofproliferation by COTI-219 plus temsirolimus. All data points aremeans±SEM (n=6). Error bars are, in every case, smaller than the size ofthe symbol.

Temsirolimus (10 nM or greater) plus 0.1 μM COTI-219 hadgreater-than-additive inhibition of proliferation. All temsirolimusconcentrations plus 0.5 or 1.0 μM COTI-219 had greater-than-additiveeffects.

In Combination with COTI-219, Rapamycin had a StronglyGreater-than-Additive Inhibitory Effect on Proliferation of U87Glioblastoma Cells

Rapamycin was prepared by crushing and extracting a tablet (suppliedfrom the LRCP Pharmacy) with DMSO, followed by centrifugation to removeparticulate matter. COTI-219 was prepared as a 50 mM stock in DMSO, thendiluted to 10 mM in DMSO followed by serial dilution water for additionto medium overlaying cells. Cells were incubated with rapamycin incombination with COTI-219 for 4 days (in wells of 96 well plates)followed by analysis of live cell density using the fluorescent vitalstain alamarBlue.

Referring to FIG. 16, relative cell density was plotted againstconcentration of COTI-219, normalized to the inhibitory effect ofrapamycin alone (this ranged from 20-30% inhibition at very lowconcentrations to 80-90% inhibition at the higher end of concentrationsused in this study). Thus, data points indicating proliferation lessthan control values indicate greater-than-additive inhibition ofproliferation by COTI-219 plus rapamycin.

All data points are means±SEM (n=6). Error bars are, in every case,smaller than the size of the symbol.

Rapamycin (5 μM or greater) plus 0.1 μM COTI-219 hadgreater-than-additive inhibition of proliferation. All rapamycinconcentrations plus 0.5 or 1.0 μM COTI-219 had greater-than-additiveeffects.

In Combination with COTI-2, Temsirolimus had a Greater-than-AdditiveInhibitory Effect on Proliferation of U87 Glioblastoma Cells

Temsirolimus was provided in liquid form from the LRCP Pharmacy. COTI-2was prepared as a 50 mM stock in DMSO, then diluted to 10 mM in DMSOfollowed by serial dilution water for addition to medium overlayingcells. Cells were incubated with temsirolimus in combination with COTI-2for 4 days (in wells of 96 well plates) followed by analysis of livecell density using the fluorescent vital stain alamarBlue.

Referring to FIG. 17, relative cell density was plotted againstconcentration of COTI-2, normalized to the inhibitory effect oftemsirolimus alone (this ranged from 20-30% inhibition at very lowconcentrations to 80-90% inhibition at the higher end of concentrationsused in this study). Thus, data points indicating proliferation lessthan control values indicate greater-than-additive inhibition ofproliferation by COTI-2 plus temsirolimus.

All data points are means±SEM (n=6). Error bars are, in every case,smaller than the size of the symbol.

Temsirolimus (100 nM or greater) plus 10 nM COTI-2 hadgreater-than-additive inhibition of proliferation. All temsirolimusconcentrations plus 25, 50, or 75 nM COTI-2 had greater-than-additiveeffects.

In Combination with COTI-2, Rapamycin had Either a Greater-than-AdditiveInhibitory Effect or a Potentially Antagonistic Effect, at HighConcentration, on Proliferation of U87 Glioblastoma Cells

Rapamycin was prepared by crushing and extracting a tablet (suppliedfrom the LRCP Pharmacy) with DMSO, followed by centrifugation to removeparticulate matter. COTI-2 was prepared as a 50 mM stock in DMSO, thendiluted to 10 mM in DMSO followed by serial dilution water for additionto medium overlaying cells. Cells were incubated with rapamycin incombination with COTI-2 for 4 days (in wells of 96 well plates) followedby analysis of live cell density using the fluorescent vital stainalamarBlue.

Referring to FIG. 18, relative cell density was plotted againstconcentration of COTI-2, normalized to the inhibitory effect ofrapamycin alone (this ranged from 20-30% inhibition at very lowconcentrations to approximately 50% inhibition at the higher end ofconcentrations). Thus, data points indicating proliferation less thancontrol values indicate greater-than-additive inhibition ofproliferation by COTI-2 plus rapamycin.

All data points are means±SEM (n=6). Error bars are, in every case,smaller than the size of the symbol.

All concentrations of rapamycin plus 10 or 25 nM COTI-2 resulted ingreater-than-additive inhibition of proliferation. High rapamycinconcentration (15 μM) may have antagonized COTI-2 when COTI-2 was usedat concentrations of 50 nM or higher. COT T-2 (50 nM or higher) hadadditive or greater-than-additive effects with rapamycin (10 μM orlower).

In conclusion, these experiments show that synergistic improvements inefficacy are obtained for combinations of mTOR-Raptor inhibitors, suchas temsirolimus and rapamycin, with mTOR-Rictor inhibitors, such asCOTI-2 and COTI-219, in the treatment of cancer, particularly cancersthat are treatable by mTOR inhibitors.

Example 16 Synergistic Effect with Cytotoxic Agents

A synergistic effect or greater than additive benefit may be observedwhen compounds described herein are administered in combination withother anti-cancer agents, in particular cytotoxic agents such ascisplatin, carboplatin and paclitaxel (see FIGS. 19-22). In FIG. 19, SHP77 cells were treated with Taxol™ at the concentrations shown or withTaxol™ plus COTI-2 (31 nM, which, as a single agent, reducesproliferation by 25%). The combination of COTI-2 and Taxol™ has agreater-than-additive effect (indicated by differences between datapoints at each concentration) where indicated by asterisks. In FIG. 20,DMS114 cells were treated with Taxol™ at the concentrations shown orwith Taxol™ plus COTI-2 (31 nM, which, as a single agent, reducesproliferation by 25%). The combination of COTI-2 and Taxol™ has agreater-than-additive effect (indicated by differences between datapoints at each concentration) where indicated by asterisks. In FIG. 21,DMS114 cells were treated with cisplatin (CDDP) at the concentrationsshown or with CDDP plus COTI-2 (31 nM, which, as a single agent, reducesproliferation by 25%). The combination of COTI-2 and CDDP has agreater-than-additive effect (indicated by differences between datapoints at each concentration) where indicated by asterisks.

In FIG. 22, SHP 77 cells were treated with cisplatin (CDDP) at theconcentrations shown or with CDDP plus COTI-2 (73 nM, which, as a singleagent, reduces proliferation by 25%). The combination of COTI-2 and CDDPhas a greater-than-additive effect (indicated by differences betweendata points at each concentration) where indicated by asterisks. In FIG.23, SHP 77 cells were treated with carboplatin at the concentrationsshown or with carboplatin plus COTI-2 (73 nM, which, as a single agent,reduces proliferation by 25%). The combination of COTI-2 and carboplatinhas a greater-than-additive effect (indicated by differences betweendata points at each concentration) where indicated by asterisks. In FIG.24, DMS114 cells were treated with carboplatin at the concentrationsshown or with carboplatin plus COTI-2 (73 nM, which, as a single agent,reduces proliferation by 25%). The combination of COTI-2 and carboplatinhas a greater-than-additive effect (indicated by differences betweendata points at each concentration) where indicated by asterisks.

In conclusion, these experiments show that synergistic improvements inefficacy are obtained for combinations of cytotoxic agents, such ascisplatin, carboplatin and paclitaxel, with mTOR-Rictor inhibitors, suchas COTI-2 and COTI-219, in the treatment of various cancer cell lines,particularly those cell lines treatable by cytotoxic agents.

Example 17 Additive Effect with Certain Anti-Cancer Agents

For the combination of COTI-2 and certain anti-cancer agents, anadditive effect is observed. As shown in FIGS. 25-28, Gemcitabine andVinorelbine had an additive effect. Gemcitabine is a pyrimidine analogantimetabolite and is not known to be an mTOR-Raptor complex inhibitor.Vinorelbine does not have a major effect on either the Akt pathway ormTOR signaling pathways. No antagonistic drug interactions wereobserved. The lack of synergy with these agents supports the earliermechanistic observations, which indicate that compounds of the presentinvention have synergy when combined with drugs that affect the Aktpathway, such as the mTOR-Raptor inhibitors, but not necessarily thosethat function by other pathways.

Example 18 In Vitro Activity of COTI-2 and COTI-219 as CombinationAgents with Erlotinib and Cetuximab

The therapeutic effects of COTI-2 and COTI-219 were evaluated as singleagents and in combinations with the commercially available anti-canceragents erlotinib (Tarceva®) and cetuximab (Erbitux®) in colorectalcancer and NSCLC cell lines (Table 21).

Methods:

Cell Lines—HT29 HCT116, H292 and H1975:

A 50-mM stock of COTI-2 or COTI-219 was formulated by dissolving anappropriate amount in DMSO. The stock concentration was further dilutedto 1 mM and added to a drug plate. In the drug plate, the 1 mM COTI-2and COTI-219 were serially diluted 10-fold in DMSO. Following dilution,2 μl of test agent was transferred to the corresponding wells of acell-containing 96-well plate with 198 μl of growth media. Tarceva®(erlotinib) was supplied by LC Labs (Woburn, Mass.). Erbitux®(cetuximab) was supplied by ImClone (New York, N.Y.). Cells were treated24 h after plating with vehicle, COTI-2 (concentrations between 0.01pM-10 pM), Tarceva® (concentrations between 0.1 pM-100 pM), or Erbitux®(concentrations between 1×10⁻¹¹ mg/ml [0.1 fM]-0.1 mg/ml [0.7 nM]). Forcombination studies, a fixed concentration of Tarceva® or Erbitux® andvarying concentrations of COTI-2 or COTI-219 were used.

Cells were grown to 70% confluency, trypsinized, counted, and seeded in96-well flat-bottom plates at a final concentration of 2.5×10³−5×10³cells/well (Day 0). Cells were allowed to incubate in growth media for24 h to allow for maximum adhesion. Treatment with COTI-2 or COTI-219 orstandard agents (Tarceva® & Erbitux®) began on Day 1 and continued for72 h without re-treatment. At the 72-h time-point, treatment-containingmedia was removed. Viable cell numbers were quantified by theCellTiter-Blue® cell viability assay, which measures the conversion ofthe indicator dye (resazurin) to resorufin, an indicator of cellviability. Experiments were repeated at least twice with the sameconcentrations to determine growth inhibitory activity. Results fromthese studies were used to calculate an IC₅₀ value (concentration ofdrug that inhibits cell growth by 50% of control) for each compound.

Cell lines-COLO-205, HCT15, and SW620:

The capacity of COTI-2 or COTI-219 to reduce tumor cell proliferation,alone or in combination with Tarceva® or Erbitux® was assessed in all 3human colon tumor cell lines. The 3 cell lines were treated as follows:

Day 1: Cells were plated in 96-well flat bottom plates (VWR,Mississauga, ON, Canada) and allowed to adhere overnight.

Day 2: Addition of COTI-2, COTI-219, Tarceva®, Erbitux®, or combinationsthereof. Stock solutions of COTI-2 and COTI-219 were prepared asdescribed above (i.e., solid material was dissolved in DMSO to yield a50 mM stock; 50 mM stock was serially diluted in DMSO to yield solutionssuch that, when 2 μl was added to wells in a 96 well plate, the desiredconcentrations of COTI compounds in a total volume of 200 μl mediasurrounding treated cells was achieved). All treated and control cellswere, therefore, exposed to a 1% DMSO concentration plus the desiredconcentration of COTI-2.

Tarceva® (erlotinib) was manufactured by LC Labs (Woburn, Mass.) andErbitux® (cetuximab) was manufactured by ImClone (New York, N.Y.). BothErbitux® and Tarceva® were provided by the London Regional CancerProgram Pharmacy. All cell lines were treated with a range of Erbitux®or Tarceva® concentrations to determine, where possible, IC₅₀ values.Erbitux® concentrations up to 0.1 mg/ml had no significant effect, as asingle agent, on any of the cell lines tested. For combination studies,therefore, 0.1 mg/ml Erbitux® was the single concentration chosen tocombine with various concentrations of COTI-2 to assess combinatorialeffects on growth. Tarceva® concentrations up to 10 μM were notsufficient to reduce growth of COLO-205 or SW620 by 50% over the 4 dayassessment of the study (i.e., not sufficient to reach an IC₅₀). Thehighest concentration of Tarceva® that did not significantly affectgrowth of each of these cell lines were: COLO 205, 10 μM Tarceva®; and,SW620, 5 μM Tarceva®. Therefore, these concentrations were chosen tocombine with various concentrations of COTI-2 or COTI-219 to assesscombinatorial effects on growth.

Combination Studies:

For combination studies, a fixed concentration of Tarceva® or Erbitux®and varying concentrations of COTI-2 and COTI-219 were used. The fixedconcentration of Tarceva® and Erbitux® used varied depending on the celllines as mentioned above. In some cell lines the IC₃₀ concentration wasused, whereas in others the highest concentration that did notsignificantly affect growth was used. After the Tarceva® or Erbitux®were added to the cells, varying concentrations of COTI-2 or COTI-219were added to generate a dose-response curve. The concentration ofCOTI-2 or COTI-219 at which 50% of the cells were inhibited was recordedas the combination IC₅₀ and is reported in Tables 22 to 25.

Results and Discussion:

Test Compounds as Single Agents:

As single agents, both COTI-2 and COTI-219 showed potent activity in allseven tumor cell lines tested. Tarceva® single agent treatment in theH292 EGFR wild-type cell line resulted in sub-micromolar activity.However, fractional activity was seen in the EGFR mutant NSCLC cellline, H1975 (Tables 22 and 24), indicating that the T790M mutation inthis cell line is conferring resistance against Tarceva®, as documentedabove. Furthermore, Tarceva® exhibited very low activity against thecolon cancer cell lines regardless of their KRAS status. No doseresponse was detected for Erbitux in this assay even atconcentrations >0.1 mg/ml (Tables 23 and 25).

Combination treatment with COTI-2 and Tarceva® resulted in a combinationindex of 1 (additive) against the H292 cell line, and a combinationindex of <1.0 (synergism) in the EGFR mutant NSCLC cell line, H1975(Table 22). These data provide strong evidence for the effectiveness ofCOTI-2 against cell lines with EGFR mutations that confer resistance totyrosine kinase inhibitors. In fact, COTI-2 increases the inhibitoryactivity of Tarceva® in combination therapy regardless of EGFR status ofthe cell lines. Furthermore, the combination of COTI-2 and Tarceva® wassynergistic (CI<1.0) for the remaining three colorectal cancer celllines (COLO-205, HCT-15, and SW620), regardless of KRAS status (Table22). Similarly, the combination treatment of COTI-219 and Tarcevaresulted in a combination index of <1.0 (synergism) regardless of theKRAS mutation status (Table 24).

Combination treatment of COTI-2 with Erbitux® resulted in positiveinteractions against all the colorectal cancer cell lines (HT29,COLO-205, HCT-15, HCT116 and SW620), indicating a combination index of<1.0 (synergism) (Table 23). Evidence indicates that the KRAS mutationstatus is a strong predictor for the outcome of Erbitux® therapy incolon cancer (103). Consequently, the data from this study provideevidence that COTI-2 in combination with Erbitux® rescues the acquiredresistance conferred by KRAS mutations. Furthermore, as a single agentCOTI-2 is highly effective against these cell lines regardless of KRASmutation status. The combination of COTI-219 and Erbitux® yieldedsimilar results, such that a synergistic activity was observed(combination index <1.0) regardless of the KRAS mutation status of thecell lines tested (Table 25).

In conclusion, both COTI-2 and COTI-219 displayed a potent single agentactivity against 7 human tumor cell lines. Combination interactionassessment showed positive combination interaction for COTI-2 andCOTI-219 in combination with Tarceva® or Erbitux®.

TABLE 21 Cell Lines. Cell line Tissue Type Histology EGFR status KRASstatus COLO-205 Colon C WT WT HT-29 Colon AC WT WT HCT-15 Colon AC WTMut (G13D) HCT-116 Colon AC WT Mut (G13D) SW620 Colon AC WT Mut (G12V)H292 NSCLC C WT WT H1975 NSCLC AC Mut WT (L858R, T790M)

TABLE 22 Mean IC₅₀ for COTI-2, Tarceva ®, and Combinations. Tissue typeTest Combination Standard CI at IC₅₀ & EGFR/ Agent COTI-2 + AgentCOTI-2 + Cell KRAS COTI-2 Tarceva ® Tarceva ® Tarceva ® Line status (nM)(nM) (nM) (nM) H292 NSCLC 3.9 70.0 20.0   1.0 EGFR - WT (Additive) H1975NSCLC 10.0 40.0 >100,000 <1.0 EGFR - (Synergism) MUT COLO- Colon 4.74.0 >10,000 <1.0 205 KRAS- WT (Synergism) HCT-15 Colon 8.3 3.8 5,100<1.0 KRAS- (Synergism) MUT SW620 Colon 165.0 90.0 >10,000 <1.0 KRAS-(Synergism) MUT

TABLE 23 Mean IC₅₀ for COTI-2, Erbitux ®, and Combinations. Tissue typeTest Combination Standard CI at IC₅₀ & EGFR/ Agent COTI-2 + AgentCOTI-2 + Cell KRAS COTI-2 Erbitux ® Erbitux ® Erbitux ® Line status (nM)(nM) (mg/ml) (nM) HT-29 Colon 200.0 6.4 >0.1 <1.0 KRAS - WT (Synergism)COLO- Colon 4.7 3.0 >0.1 <1.0 205 KRAS - WT (Synergism) HCT-15 Colon 8.34.0 >0.1 <1.0 KRAS - (Synergism) MUT HCT- Colon 3.6 4.1 >0.1 <1.0 116KRAS - (Synergism) MUT SW620 Colon 165.0 82.0 >0.1 <1.0 KRAS -(Synergism) MUT

TABLE 24 Mean IC₅₀ for COTI-219, Tarceva ®, and Combinations. TestTissue type Agent Combination Standard CI at IC₅₀ & EGFR/ COTI-COTI-219 + Agent COTI-219 + Cell KRAS 219 Tarceva ® Tarceva ® Tarceva ®Line status (nM) (nM) (nM) (nM) H292 NSCLC 20.0 100.0 20.0 <1.0 EGFR -WT (Synergism) H1975 NSCLC 100.0 700.0 >100,000 <1.0 EGFR - (Synergism)MUT COLO- Colon 4.3 2.0 >10,000 <1.0 205 KRAS- WT (Synergism) HCT-15Colon 5.1 2.4 5,100 <1.0 KRAS- (Synergism) MUT SW620 Colon 7.43.1 >10,000 <1.0 KRAS- (Synergism) MUT

TABLE 25 Mean IC₅₀ for COTI-219, Erbitux ®, and Combinations. TestTissue type Agent Combination Standard CI at IC₅₀ & EGFR/ COTI-COTI-219 + Agent COTI-219 + Cell KRAS 219 Erbitux ® Erbitux ® Erbitux ®Line status (nM) (nM) (mg/ml) (nM) HT-29 Colon 7.4 30.0 >0.1 <1.0 KRAS -WT (Synergism) COLO- Colon 4.3 2.0 >0.1 <1.0 205 KRAS - WT (Synergism)HCT-15 Colon 5.1 3.1 >0.1 <1.0 KRAS - (Synergism) MUT HCT- Colon 8.7300.0 >0.1 <1.0 116 KRAS - (Synergism) MUT SW620 Colon 7.4 2.8 >0.1 <1.0KRAS - (Synergism) MUTCombination indexes (C1s) are described by Chou and Talalay (TrendsPharmacol. Sci. 4, 450-454), which is incorporated herein by reference.The CI, a numerical value calculated as described in equation A, alsoprovides a quantitative measure of the extent of drug interaction. A CIof less than, equal to, and more than 1 indicates synergy, additivity,and antagonism, respectively.CI=(C _(Ax) /IC _(x,A))+(C _(B,x) /IC _(x,B))  Equation A:Where C_(A,x) and C_(B,x), are the concentrations of drug A and drug Bused in combination to achieve x % drug effect. IC_(x,A) and IC_(x,B)are the concentrations for single agents to achieve the same effect.

Example 19 Akt as a Biomarker for COTI-2 Efficacy

It was previously demonstrated that COTI-2 interferes with the functionof AKT in SCLC cell lines, by down-regulating AKT transcription as wellas producing a functional impairment of AKT kinase activity. Therefore,the aim of the current set of experiments was to determine the extent ofthe correlation between the expression of total AKT, its isoforms (AKT1,2, & 3), and susceptibility to COTI-2 apoptosis.

Methods:

Tissue Culture:

Cell cultures were maintained under regular tissue culture conditionsand passaged 1-2 times a week as recommended by ATCC. For siRNAexperiments the manufacturer's conditions for growth and transfectionwere followed. All siRNA reagents for AKT and AKT2 transfections wereobtained from Cell Signaling.

Cells were treated for 48 h with 0-100 ng of AKT or AKT2 siRNA beforeincubation with COTI-2 at various concentrations (0-500 nM). Followingincubation for 48 h, cell viability was determined by MTT assay.

Western Blotting:

Routine laboratory methods were employed for the production of cellularlysates, protein quantification, SDS-PAGE and western blotting. Allantibodies were obtained from Cell Signaling and Abcam. To determinetotal AKT & AKT2 in DMS114 cells, 5×10⁴ cells were incubated for 48 hwith 10-100 ng of AKT2 siRNA or a scrambled control oligonucleotide.Cells were lysed and 50 μg of protein was separated by SDS-PAGE andblotted with antibody to total AKT or AKT2.

Results and Discussion:

The expression of AKT1, AKT2, AKT3, total AKT and β-actin were examinedin a panel of cell lines representing various types of cancers, with theaim of determining if a correlation exists with the pre-determinedIC₅₀'s for these cell lines. As shown in FIG. 33, when corrected fortotal AKT expression, only AKT2 expression was statisticallysignificantly and inversely correlated to the COTI-2IC50 for this panelof cell lines. In order to confirm that AKT2 expression is needed forCOTI-2 induced apoptosis, DMS114 and SHP77 cell lines were treated for48 h with 0-100 ng of AKT or AKT2 siRNA before incubation with 500 nM ofCOTI-2 (FIG. 34). Both total AKT and AKT2 siRNA in the absence of COTI-2caused significant apoptosis of DMS114 cells indicating that the siRNAfunctions as expected. The viability of DMS114 cells incubated with AKTsiRNA in the presence or absence of COTI-2 is unchanged, thus indicatingthat AKT and more specifically AKT2 is the cellular target of COTI2. IfCOTI-2 had an alternate target, one would expect differential cell deathin the presence and absence of COTI-2. The down-regulation of AKT2 priorto the addition of COTI-2 was confirmed with western blotting in DMS114cell lines using an antibody to AKT2 (FIG. 35). Overall, these dataindicate that in the absence of its cellular target (AKT2) which wasknocked-down by siRNA specific for AKT2, COTI-2 was unable to exertadditional significant cytotoxicity on the cell lines.

Similar results were obtained with SHP77 cell lines, such that nosignificant difference in cell death was observed when the cells weretreated with total AKT or AKT2 siRNA in the presence or absence ofCOTI-2 (FIG. 36). Therefore, total AKT and AKT2 siRNA abrogates COTI-2induced apoptosis in SHP77 cells. Furthermore, transfection with 100 nMof AKT2 siRNA, but not with total AKT siRNA, caused significantapoptosis in SHP77 cell lines (FIG. 37).

The phospho-AKT status at Ser473 was determined in the presence ofCOTI-2 utilizing western blotting. A profound inhibition of AKT Ser473phosphorylation is noticeable at 2 h, the earliest time-point followingincubation with 250 nM COTI2 (FIG. 38). These data were confirmed using2 different antibody preparations from Cell signaling and Abcam. Theinhibition of AKT phosphorylation at Ser473 correlates with thereduction in the phosphorylation of GSK-3α/β (pSer21/9), which is adownstream target of activated phospho-AKT. Therefore, these dataindicate that AKT is the cellular target of COTI-2, since COTI-2prevents phospho-activation of AKT and consequently AKT is unable tophospho-inhibit its downstream target, GSK-3α/β at Ser21/9. The completetime course of the AKT phospho-Ser473 status in DMS114 and SHP77 cellsduring incubation with COTI-2 is documented in FIG. 39.

A complex relationship exists between Raptor- and Rictor-containing mTorcomplexes mTORC1 and mTORC2, respectively, and AKT. mTORC2 activates AKTby Ser473 phosphorylation; in turn, activated AKT phosphorylates TSC2,which prevents its interaction with Rheb and thus the hydrolysis of GTPassociated with Rheb. As a result, Rheb-GTP activates mTOR byphosphorylation on Ser2448. These events suggest that COTI-2, byinhibiting the formation of active phospho-Ser473 AKT, would also leadto a decrease in mTOR phospho-Ser2448 phosphorylation, as less Rheb-GTPbecomes available, due to its increased sequestration in complexes withTSC2. The results shown in FIGS. 40-45 agree with this hypothesis. InDMS114 cells incubated with COTI-2 there is an initial decrease inphospho-Ser 2448 at 2 hrs followed by a more sustained decrease at 24and 48 hrs. In SHP77 cells, only the decrease at 2 hrs of incubation wasobserved. Interestingly, in both cell lines there is a delayed effectthat impairs the autophosphorylation of mTOR on Ser2481. This event wasshown to be wortmannin sensitive but a direct connection betweeninhibition of PI3K and impaired mTOR phospho-Ser2481 has not beendemonstrated (J Biol Chem, 275: 7416-7423). Surprisingly, a strong buttransient increase in PTEN phospho-Ser380 was observed in DMS114 cells.Phosphorylation of the PTEN C terminal was reported to stabilize PTENand protect against ubiquitin-mediated degradation (Nat Rev Cancer, 6:184-192). One implication of this observation is that COTI-2 mayactivate PTEN and thus, by reducing the amount of PIP3, contribute tothe inhibition of AKT activity.

These experimental results provide yet further evidence that thecellular target of COTI-2 is AKT, specifically AKT2, and that COTI-2 islikely implicated in mTOR-Rictor complex formation.

Example 20 PTEN siRNA Knockdown in Human Tumor Cell Lines (HeLa andMCF-7) Results in Increased Resistance to COTI-2

The activation of Akt is negatively regulated by the tumor suppressorPTEN (phosphatase with tensin homology), which is a lipid phosphatase,therefore, inactivation of PTEN by siRNA results in increased levels ofactive Akt. In order to further investigate whether Akt is the likelytarget of COTI-2, the susceptibility to killing by COTI-2 was evaluatedin 2 human tumor cell lines (HeLa and MCF-7) transfected with PTENsiRNA. Previous work has demonstrated that increased PTEN activityreduces Akt activity, and knockdown of PTEN has been shown to increaseAkt activity in human tumour cells (Cancer Cell, 12:395-402). Therefore,if Akt is the target for COTI-2, then it is expected that increasedlevels of Akt resulting from PTEN siRNA inactivation will causeincreased resistance to COTI-2. Transfected cells were confirmed bywestern blot analysis and reverse transcription PCR were grown andproliferation assessed in the presence or absence of COTI-2.

Methods:

Briefly, HeLa and MCF-7 cells were transfected with 100 nM PTEN siRNA orcontrol non-targeted siRNA at 48 or 144 h prior to exposure to variousconcentrations of COTI-2.

Western Blot Analysis:

Tranfected HeLa cells were confirmed by western analysis as described.Western blot analysis of extracts from HeLa cells transfected withnon-targeted (−) or targeted (+) siRNA (Cell Signaling Technologies,Inc., SignalSilence PTEN siRNA, human-specific; and control,non-targeted SignalSilence siRNA from the same manufacturer). PTEN wasdetected using the PTEN Antibody #9552, and p42 was detected using thep42 MAPK Antibody #9108. The PTEN Antibody confirms silencing of PTENexpression, and the p42 MAPK Antibody is used to control for loading andspecificity of PTEN siRNA (data obtained from Cell Signaling Technologywebsite, http://www.cellsignal.com/products/6251.html). Since PTEN siRNAwas capable of downregulating PTEN protein in HeLa cells (supported bydata generated by Cell Signaling Technologies, Inc.), PTEN siRNA andnon-targeting control siRNA used for the experiments described belowwere obtained from New England Biolabs Ltd. (Ontario, Canada).

Effect of COTI-2 on Cell Proliferation:

To assess the capacity of transient siRNA transfection to alter theability of cells to proliferate in the presence or absence of COTI-2,HeLa or MCF-7 cells (both PTEN-positive) were plated in T25 flasks andtransfected 24 h later with PTEN siRNA or non-targeting control siRNA(100 nM in both cases), using Oligofectamine Reagent (Invitrogen)according to the manufacturer's instructions and as previously described(J Pharmacol Exp Ther, 322:123-132). In brief, siRNA:OligofectamineReagent complexes were formed in DMEM without FBS, then serially dilutedto generate desired transfection concentrations. siRNA complexes in DMEMwere added to HeLa or MCF-7 cells grown to 50 to 60% confluence. PTENand 18S rRNA mRNA levels were determined by semi-quantitative reversetranscription PCR. PTEN primers were: PTEN forward(5′CCACCACAGCTAGAACTTATC3′; PTEN reverse (5′ATCTGCACGCTCTATACTGC3′).GAPDH primers were as described previously (J Pharmacol Exp Ther,322:123-132). The reverse transcription/PCR method was as described inthe same publication. The ratio of PTEN mRNA/18S rRNA for untransfectedcells was normalized to 100% and all other PTEN mRNA/18S rRNA ratioswere reported as a percent of that 100% value.

For determination of drug sensitivity, HeLa and MCF-7 cells (transfectedwith PTEN siRNA or control, non-targeted siRNA 48 or 144 hours prior)were re-plated in 96-well plates (VWR, Mississauga, ON, Canada) at 1700(MCF-7) and 1200 (HeLa) cells per well in a volume of 100 μl of DMEM+10%FBS. Cells were allowed to adhere to tissue culture plastic for 18 hbefore addition of COTI-2M05 at various concentrations. COTI-2 was addedin a volume of 100 μl (from a stock DMSO/DMEM solution) so that thefinal DMSO concentration on cells was less than 0.1%. Cells were thengrown for 4 days, and viable cell numbers were assessed by the alamarBlue fluorescence assay using a Wallac Victor² multilabel counter(PerkinElmer Wallac, Gaithersburg, Md.). For each concentration ofCOTI-2M05, proliferation was defined as the “number of viable cellstransfected with PTEN siRNA, expressed as a % of the number of viablecells transfected with control, non-targeting siRNA”. This calculationmethod corrected for minor effects of PTEN knockdown alone on growthrate and/or cell viability.

Statistical Analysis:

Data are presented as means±S.E. To determine the significance ofdifferences between means, a Student's t test was performed. The levelof significance for all statistical analyses was chosen a priori to bep<0.05.

Results and Discussion:

Western blot analysis of HeLa transfected with targeted (+) andnon-targeted (−) siRNA confirm that the PTEN siRNA specificallyknocks-down the expression of PTEN (FIG. 44). Furthermore, reversetranscription PCR analysis indicated that PTEN siRNA transfectionknocked down PTEN mRNA levels by a maximum of approximately 90% in HeLacells (FIG. 45A) and 70% in MCF-7 cells (FIG. 45B), by 48 hpost-transfection. By 114 h (6 days post-transfection), PTEN levels hadrecovered to control values in both HeLa (FIG. 45A) and MCF-7 (FIG. 45B)cells (in accord with the capacity the mRNA levels of the cells torecover from targeting siRNAs). These data show that the transfection ofboth cells lines with PTEN siRNA was specific and by 114 h theinhibitory effect of the PTEN siRNA no longer exists.

The transfected cells were then analyzed for their susceptibility tokilling by COTI-2 using the alamar Blue fluorescence assay describedpreviously. The transfection of cells with PTEN siRNA resulted in asignificant increase in the resistance of both cell lines (HeLa andMCF-7) to COTI-2 at 48 h post-transfection (FIGS. 46A & 46B,respectively). By 144 h post-transfection there is no significantdifference in the killing of cells transfected with PTEN siRNA ornon-targeted siRNA. These data are consistent with the recovery ofnormal PTEN mRNA levels by 144 h (refer to FIGS. 45A and 45B). Theincreased resistance to COTI-2 in the 2 PTEN-positive human tumor celllines (HeLa and MCF-7) after siRNA knockdown of PTEN and presumedincreased Akt activity is consistent with the hypothesis that Akt is adirect or indirect target of COTI-2 in human tumor cells. The multipleconfirmations of Akt, and particularly Akt2, as the molecular target ofthe compounds of the present invention, particularly COTI-2, indicatethat these compounds should be effective in the treatment of cancerscharacterized by over-expression of Akt. Compounds of the presentinvention are presumed to be efficacious in the treatment of all cancerscharacterized by overexpression of Akt, in particular those cancers inaddition to lung cancer, cervical cancer, ovarian cancer, cancer of CNS,skin cancer, prostate cancer, sarcoma, breast cancer, leukemia,colorectal cancer, head cancer, neck cancer or kidney cancer, which arealready listed above.

Example 21 Sensitivity of Human Tumour Cell Lines (H226 NSCLC and HL-60Promyelocytic Leukemia) and Normal Primary Peripheral Blood MononuclearCells (PBMCs) to COTI-2

The IC₅₀ of COTI2-M05 was determined against normal human peripheralblood mononuclear cells (PBMCs) as an indication of the toxicity of thecompound against healthy non-cancerous cells. Both human promyelocticleukemia human (HL-60) and human NSCLC (H226) cell lines were used as acomparator against the PBMCs.

Methods:

All cell lines were grown and exposed to various concentrations ofCOTI-2 or vehicle only control and the percentage of dead or dying cellswere determined 24 and 48 h post-exposure by Annexin V and propidiumiodide.

Tumour Cell Lines and Primary Non-Tumour Human Peripheral BloodMononuclear Cells:

HL-60 (human promyelocytic leukemia) cells were grown as non-adherentcells in flasks in RPMI 1640 medium supplemented with 10% FBS. H226(human non-small cell lung cancer) cells were grown in Eagle's MEMsupplemented with 10% FBS. Both cell lines were obtained from theAmerican Type Culture Collection (ATCC)(Rockville, Md.) and maintainedat 37° C. in a humidified 5% CO₂ atmosphere.

Blood samples from 2 healthy human volunteers (male, 22 years of age;female, 23 years of age) were obtained in 5 ml Vacutainers® containing100 USP sodium heparin (Becton Dickinson, Franklin Lake, N.J.) androcked gently to mix, then kept at room temperature. Blood was diluted1:1 with phosphate buffered saline (PBS) and layered over an equalvolume of room temperature Histopaque-1083® (Sigma Diagnostics, St.Louis, Mo.) in a 15 ml conical bottom polyethylene (PET) centrifuge tube(Corning Corporation, Corning, N.Y.). This was centrifuged at 400×g for30 minutes at 20° C. (no brake), to allow separation of mononuclearcells from whole blood. The serum layer was removed by Pasteur pipetteand discarded. The mononuclear cell layer at the top of the Histopaquewas removed by Pasteur pipette and placed in 15 ml PET centrifuge tube.Cells were washed twice with 10 ml PBS, with centrifugation at 250×g for15 minutes (maximum brake) to precipitate cells away from the PBS washfluid. The pellet was suspended in 37° C. RPMI 1640 media (Gibco BRL,Grand Island, N.Y.) with or without 10% fetal bovine serum (Gibco BRL,Grand Island, N.Y.), depending on experimental requirements.

Flow Cytometric Analysis of Cell Death:

Cells were plated in 25-cm² flasks and treated with COTI-2 (0-100 μM)added from a stock DMSO/DMEM solution such that DMSO concentrationsnever exceeded 0.5%. Control cells were treated with stock DMSO/DMEM. At24 and 48 h after addition of drug or control (vehicle alone),supernatant medium (containing all HL-60 cells or any non-adherent H226cells or non-tumour primary peripheral blood mononuclear cells) wascollected into 13 ml conical centrifuge tubes. Remaining adherent cellswere rinsed with ice-cold PBS, trypsinized, and added to thenon-adherent fraction. Cells were centrifuged (100×g for 10 min at 4°C.), washed once more in ice-cold PBS, re-precipitated bycentrifugation, and re-suspended (1000 cells per μl) in binding buffer(140 mM NaCl₂,2.5 mM CaCl₂, 10 mM HEPES). Resuspended cells (100 μl)were incubated in the dark with 10 μl propidium iodide (PI, 50 ng perml; Sigma, St. Louis, Mo.) plus Annexin V-FITC (25 ng per ml, greenfluorescence; BD Biosciences, Mississauga, Ontario). The AnnexinV-positive cells indicate dying or apoptotic cells and dead cells,whereas the propidium iodide stains for dead cells. Samples wereanalyzed for combined Annexin V/PI staining (to assess dead plusapoptotic cells) using a Beckman Coulter EPCS XL-MCL flow cytometer(Beckman Coulter, Hileah, Fla.) and the data were analyzed withCellQuest software.

Results and Discussion:

The H226 promyeloctic leukemia cell lines were chosen for this studysince they exhibited the least sensitivity of the panel of human tumorcell lines previously tested for sensitivity to growth inhibition byCOTI-2. The induction of apoptosis and death in approximately 40% ofthese cells at 48 h after treatment with 15 μM of COTI2-M05 isconsistent with the previously determined IC₅₀ (FIG. 47). Similarly,induction of greater than 30% death at 24 h and greater than 70% deathat 48 h in HL-60 cells after treatment with 200 nM of COTI-2 isconsistent with our previous report of an IC₅₀ for HL-60 cells of 236 nM(±9) (FIG. 47).

PBMCs from healthy adult human male and female subjects were more than500-fold less sensitive than HL-60 cells (derived from human monocytes)to death induction by COTI2-M05 (FIG. 47). The healthy PBMCs wereexposed to COTI2-M05 at a concentration of 100 μM, which is the maximumpractical concentration to which these normal mononuclear cells could beexposed. At this concentration, COTI-2 induced some cell death in maleand female PBMCs compared to vehicle only controls, however, thisconcentration was less than the IC₅₀ of these cells (i.e., IC₅₀>100 μM).Furthermore, compared to H226 cells, normal PBMCs were 6- to 7-fold lesssensitive to death induction by COTI-2 (FIG. 47).

In conclusion, these experiments indicate that normal differentiatedhuman cells were much less sensitive than human tumor cells to apoptoticdeath by COTI2-M05. One limitation of these experiments is thatimmortalized human tumor cell lines (HL-60 & H226) capable of ex vivoproliferation were compared against non-immortalized human PBMCs thatare not capable of ex vivo growth under the conditions used.Interestingly, despite this limitation, we see a significant differencein cell death in tumor cell lines relative to non-tumor healthy cells.

Example 22 Molecular Target Validation for COTI-219 in SCLC Cell Lines

Gene expression profile/MPM analysis of the molecular pathwaysresponsible for COTI-2,9-induced apoptosis in small cell lung cancer(SCLC) cell lines identified several potential targets for thiscompound. The principal aim of the experiments described here was toprovide validation for these targets and further help define themechanism of action of COTI-219. siRNA technology was used to knock-downRas, Bim, CDK2, Egfr, Erbb2, Erk1/2, and PKC-α, in 2 SCLC cell lines,namely DMS114 and SHP77.

Methods:

In brief, cells DMS114 and SHP77 cells were transfected with genespecific siRNA in the presence or absence of COTI-219 and cell viabilitydetermined after 24 to 48 h post-incubation using the trypan blueexclusion method.

Tissue Culture and siRNA Experiments:

Cell cultures were maintained under regular tissue culture conditionsand passaged 1-2 times a week as recommended by ATCC. With the exceptionof the siRNA for Bim (Signal Silence Bim siRNA kit #6460 from CellSignaling), all other siRNA reagents for gene knock-down experiments wasobtained from Ambion (Silencer Negative Control #1 siRNA #AM4611, CDK2Silencer Select #s204, HRAS Silencer Select #s807, Protein kinase Calpha Silencer Validated #301, MAPK3/Erk Silencer Pre-designed #214749,EGFR Silencer Select #s563, and ERBB2 Silencer Select #s611). Initially,the manufacturer's suggested conditions for transfection were followedbut the extent of knock-down obtained was in some cases poor andunsuitable for drug target validation experiments. The transfectionmethod required optimization such that a knock-down of the target genegreater than ˜75% was achieved and confirmed by western blot analyses.In some cases, siRNA concentrations up to 100 nM were required toproduce a useful degree of target knock-down. Furthermore, for somesiRNA's useful target knock-downs were achieved by forward transfectionprotocols while for others reverse transfection was necessary. Forforward transfection protocols, up to 2.0×10⁵ cells were platedovernight, followed by incubation with the siRNA reagent for anadditional 48 hrs. In reverse transfection protocols ˜0.1×10⁵ cells wereincubated with the siRNA reagent at the time of plating, omitting the 24h pre-plating step. For each siRNA used in the gene knock-downexperiments the transfection protocol was optimized with respect to thenumber of cells, amount of siRNA and transfection method so that aknock-down of the target gene >75% was achieved. Following an optimalknock-down level at >75%, 2×10⁵ cells were incubated with 500 nMCOTI-219 and number of viable cells was measured at 48 h.

Cell Viability and Western Blotting:

Cell viability assays were performed 24 to 48 h following incubationwith COTI-219 by the trypan blue exclusion method using aBeckman-Coulter Vi-CELL XR 2.03 automated cell counter. Routinelaboratory methods were employed for the production of cellular lysates,protein quantitation, SDS-PAGE and western blotting. All antibodies wereobtained from Cell Signaling (Erk1/2 #4695, Ras #3965, CDK2 #2546,PKCalpha #2056, EGFR #2232, and ERBB2 #2165). A Student's T-test wasused to compare control and COTI-219 treated cells in order to determinea significant difference (p<0.01).

Cell Cycle Analysis:

Approximately 2×10⁵ DMS114 cells were incubated for 24 h with theindicated concentrations of COTI-219. Following incubation with COTI-219the cells were washed and re-suspended in 0.5 ml of BP PharmingenPropidium Iodide staining solution (Cat. No. 556463) for FACS analysisin order to determine the changes in the cell cycle.

Results and Discussion:

The transfection method required optimization such that a knock-down ofthe target gene greater than ˜75% was achieved and confirmed by westernblot analyses. Representative western blot analysis of DMS114 and SHP77cells transfected with gene-specific siRNA or scrambled control siRNAafter 48 h incubation are illustrated in FIG. 48 in order to demonstratethe extent of down-regulation of target genes obtained. Notably, Egfrand Erbb2 expression was very low in DMS114 cells, but SHP 77 cellsexpress robust levels of these proteins.

FIG. 48 shows representative western blot analysis of DMS114 and SHP77cells transfected with gene-specific siRNA or scrambled controloligonucleotide for 48 h. Actin control blots (not shown) demonstratedthat similar amounts of protein were loaded. SC-CT, scrambled controloligonucleotide; D, DMS114; S, SHP77; f, forward and r, reversetransfection protocols.

Following optimization of siRNA transfection, cells were incubated for48 h with 500 nM COTI-219 and cell viability determined. In DMS114 cellsonly the Ras knock-down was able to suppress the action of COTI-219 onapoptosis/cell growth (FIG. 49A). The fact that knock-down of Erbb2significantly decreases cell viability even in the absence of COTI-219is surprising given than Erbb2 protein levels were barely detectable inthis cell line. It is unclear if this represents a non-specific event.Unlike DMS114, SHP77 cells express robust levels of Egfr and Erbb2. Inthis cell line, not only Ras but Erbb2, Erk1, and PKC-α all suppressedthe apoptotic/cell growth inhibitory effect of COTI-219 (FIG. 49B). Apossible explanation is that Erbb2, PKC-α, and Erk1/2 knock-downs arelikely to slow-down cellular proliferation by inhibiting cyclin D1activity. Cyclin D1 mRNA is only moderately expressed in SHP77 cells asopposed to non-small cell lung cancer cells, where gene amplificationhas been reported. As a result, Erbb2 and its downstream effectors,including PKC-α and Erk1/2 may play a significant role in cyclin D1up-regulation. In the absence of sustained levels of Erbb2, PKC-α, orErk1/2 to regulate cyclin D1 expression the ability of COTI-219 toinduce G1 arrest and apoptosis may be impaired. Since the cell viabilityassay reflects the compounded effects of apoptosis and growth inhibitionit cannot differentiate between the relative effects of the knock-downon the two processes. Furthermore, COTI-219 induces both apoptosis andcycle arrest at the concentration used in these experiments (FIG.49A-B). Therefore, while knockdown of Ras likely suppresses COTI-219apoptosis, the knock-down of Erbb2, may suppress the growth inhibitoryeffects of COTI-219.

In lung cancer, oncogenic Ras exerts anti-apoptotic effects, in part byup-regulating the multi-functional PKB/AKT kinase. Activated PKB/AKTphosphorylates and inhibits caspase 9 activation. In order to determinewhether caspase 9 activation mediates COTI-219 apoptosis, cells wereincubated for 12 h with vehicle or increasing concentrations of COTI-219and the expression levels of cleaved caspases 3 and 9 were determined.There was a dose-dependent increase in the levels of both cleavedcaspases, peaking at 0.5 μM of COTI-219. Beyond this concentration,activated caspase 9 was not detectable and there was a decline incleaved caspase 3. These results demonstrated that COTI-219 can induceapoptosis by a different mechanism(s), which is dose-dependent.

Cells were incubated with increasing concentrations of COTI-219 and cellcycle analyzed in order to further investigate the mechanism of actionof COTI-219 since at concentrations >0.5 μM there was no detectablecaspase 9 cleavage (FIG. 50). Control cells displayed the characteristicpattern of stained nuclei in the Go/G1/S/G2 stages of the cell cycle(FIG. 51). However, incubation with 1.5 μM of COTI-219 produced cellcycle arrest in G1. At higher doses of COTI-219 cells escape G1 arrestand the cell cycle appears to be normal. These observations furtherstrengthen the observation that different mechanisms are responsible forCOTI-219 apoptosis, depending upon the concentration of the compound.

The role of the pro-apoptotic regulator Bim1 remains uncertain. Althoughit has been reported that small molecule inhibitors of the Egfr/Erbb2pathway can induce apoptosis by up-regulating Bim1 expression, andconsequently sequestering anti-apoptotic Bcl2, knock-down of Bim did notinterfere with COTI-219 apoptosis. However, cells that survived COTI-219apoptosis had significantly lower levels of Bim than did control cells(not shown), suggesting that Bim1 may play a role in the observedeffects of COTI-219.

In conclusion the cumulative data presented in this study suggest that,at nanomolar ranges of concentrations (≦500 nM), COTI219 inducesapoptosis through its inhibitory effect on Ras; whereas, at highermicromolar concentrations (≧1.5 μM), COTI219 induces cell cycle arrestthrough an unidentified target. This concentration of COTI-219 isimportant in understanding the mechanism of action of COTI-219, ashigher concentrations may affect a different set of molecular targetsand induce apoptosis by different mechanisms (see further).

Example 23 In Vitro Pharmacokinetics (ADME Toxicology)

The pharmacokinetics of many antineoplastic agents are associated withtreatment outcome, which makes it desirable to evaluate these parametersin various in vitro and in vivo screens (Nat Rev Cancer 5: 447-458).ADMET, which is an acronym for absorption, distribution, metabolism,excretion and toxicity, describes the disposition of a pharmaceuticalcompound within an organism (Nat Rev Cancer 6: 546-558). These criteriaall influence the drug levels and kinetics of exposure to the tissuesand hence influence the performance and pharmacological activity of thecompound. An ensemble of tests is used to characterize a compound'sproperties with respect to the criteria listed above. Consequently,COTI-2 was evaluated for its plasma protein binding, absorption (Pgpsubstrate/inhibitor), metabolic stability, and CYP450 inhibitionpotential using in vitro experiments to characterize itspharmacokinetics.

Methods:

Plasma Protein Binding:

The experimental method employed which was described previously (J PharmSci 92: 967-974) is outlined below.

Test Analytical Assay Compound Equilibration/Incubation Method PlasmaProtein 10 μM (n = 2) At least 8 hours at 37° C. HPLC- Binding 1% DMSOin human plasma 12-14K MS/MS (human) MWCO dialysis membrane 0.05 Mphosphate buffer, pH 7.5 Plasma Protein 10 μM (n = 2) At least 8 hoursat 37° C. HPLC-MS/ Binding (rat, 1% DMSO in rat plasma 12-14K MS mixedbreeds) MWCO dialysis membrane 0.05 M phosphate buffer, pH 7.5 PlasmaProtein 10 μM (n = 2) At least 8 hours at 37° C. HPLC-MS/ Binding (dog,1% DMSO in dog plasma 12-14K MS mixed breeds) MWCO dialysis membrane0.05 M phosphate buffer, pH 7.5 Notes: 96-well dialysis apparatus: fromHTDialysis LLC (Gates Ferry, CT), part #1006 Abbreviations: DMSO:Dimethylsulfoxide HPLC-MS/MS: HPLC coupled with tandem mass spectrometry(Instrumentation: Thermo Finnigan) HPLC: High performance liquidchromatrography MWCO: Molecular weight cut-off

Commercially obtained (Rockland Immunochemicals Inc.), non-sterile,species-specific plasma (human, monkey, and dog) with sodiumanticoagulant added was utilized for this study. An equilibrium dialysisexperiment was performed in a 96-well format in a dialysis blockconstructed from Teflon (J Pharm Sci 92: 967-974). Dialysis membranestrips (12-14K MWCO) were presoaked in water and ethanol, rinsed andthen kept in buffer until use. Following assembly of the 96-welldialysis apparatus, 0.15 ml of the dialysis block was assembled andmembrane strips placed between the dialysis and sample compartments. Thedialysate compartment was loaded with 0.15 ml phosphate buffer was addedto the dialysis side of each well. Plasma (unfiltered) was spiked with10 μM COTI-2 (1% DMSO) and 0.15 ml added to the sample side of eachwell. After loading, samples are covered and incubated at 37° C. untilequilibrium was reached (at least 8 hours). Equal volumes of sample wereremoved from the buffer and plasma sides of each well, diluted withacetonitrile/buffer and centrifuged. Also at this time an additionalsample was prepared (in duplicate) by spiking COTI-2 in plasma at 10 μM,followed by sampling and diluting in acetonitrile/buffer in the samemanner as the incubated plasma sample. This calibration sample served asthe basis of a recovery determination. The supernatants were analyzed byHPLC-MS/MS and the peak response of COTI-2 in both dialysis and samplecompartments, and in the calibration sample, were subsequentlydetermined in two independent replicates. Acebutolol, quinidine andwarfarin were included in each assay as reference compounds. Thereference compounds yield protein binding values that represent low,medium and high binding to plasma proteins, respectively. Samples wereanalyzed via (RP)HPLC-MS/MS using selected reaction monitoring (SRM).The HPLC conditions consist of a binary LC pump with autosampler, a C18column (2×20 mm), and gradient elution. The percent bound toplasma/serum proteins and percent recovery of the compounds werecalculated. The recovery determination serves as an indicator as to thereliability of the calculated protein binding value.

In Vitro Absorption (Pgp Substrate or Inhibitor):

The general procedures are outlined below.

Test Biological Assay Concentration Conditions Analytical Methods A-BPermeability 10 μM in HBSS A-to-B flux at 37° C. HPLC-MS/MS (TC7, pH7.4/7.4) 1% DMSO (n = 2) with shaking 96-well Multiscreen plate pH 7.4in A and pH 7.4 in B Donor samples: time 0 and 60 min Receiver samples:time 60 min A-B Permeability 10 μM in HBSS A-to-B flux at 37° C.HPLC-MS/MS (TC7, pH 7.4/7.4) + 100 μM with shaking 96-well verapamilverapamil in A Multiscreen plate and B sides, 1% pH 7.4 in A and pH DMSO(n = 2) 7.4 in B Donor samples: time 0 and 60 min Receiver samples: time60 min B-A Permeability 10 μM in HBSS B-to-A flux at 37° C. HPLC-MS/MS(TC7, pH 7.4/7.4) 1% DMSO (n = 2) with shaking 96-well Multiscreen platepH 7.4 in A and pH 7.4 in B Donor samples: time 0 and 40 min Receiversamples: time 40 min B-A Permeability 10 μM in HBSS B-to-A flux at 37°C. HPLC-MS/MS (TC7, pH 7.4/7.4) + 100 μM with shaking 96-well verapamilverapamil in A Multiscreen plate and B sides, 1% pH 6.5 in A and pH DMSO(n = 2) 7.4 in B Donor samples: time 0 and 40 min Receiver samples: time40 min P-glycoprotein Test compound B-to-A flux at 37° C. Scintillationinhibitation (0.03, 0.3, 1, 3, 5, with shaking 96-well counting (TC7,³H-digoxin 10, 30 and 100 μM Multiscreen plate substrate) in both A andpH 7.4 in A and B B sides) ³H- sides digoxin in B side, Donor samples:digoxin (10 μM) time 180 min in A side and B Receiver samples: side inHBSS, time 180 min 0.1% BSA, 1% DMSO (n = 2) Notes: Multiscreen plate:96-well plate, from Millipore, catalog number MACACO2S5 Abbreviations:A: Apical side B: Basolateral side DMSO: Dimethylsulfoxide HBSS: Hank'sbalanced salt solution, from Invitrogen, catalog number 14065-056, plus5 mM HEPES, from Sigma, catalog number H 3375, pH 7.4 HEPES:N-(2-hydroxyethyl)-piperazine-N′-(2-ethanesulfonic acid) HPLC-MS/MS:HPLC coupled with tandem mass spectrometry (Instrumentation: ThermoFinnigan) HPLC: High performance liquid chromatrography

Passage Days in Reference Bib- Assay Cell Number Culture Compoundliography A-B TC7 15 13 to 25 4 reference Gres et al. Permeability(human passages compounds (1998) (TC7, pH intestinal in culture (set 2)7.4/7.4) epithelial between cells) passages 20 and 40 A-B TC7 15 13 to25 Gres et al. Permeability (human passages (1998) (TC7, pH intestinalin culture 7.4/7.4) + epithelial between verapamil cells) passages 20and 40 B-A TC7 15 13 to 25 4 reference Horio M. Permeability (humanpassages compounds et al. (TC7, pH intestinal in culture (set 2) (1989)7.4/7.4) epithelial between cells) passages 20 and 40 B-A TC7 15 13 to25 Hunter et Permeability (human passages al. (1993) (TC7, pH intestinalin culture 7.4/7.4) + epithelial between verapamil cells) passages 20and 40 P- TC7 15 13 to 25 Verapamil Cavet et glycoprotein (humanpassages al. (1996) inhibition intestinal in culture (TC7, ³H-epithelial between digoxin cells) passages substrate) 20 and 40 Notes:TC7 is a sub-clone of the Caco-2 cell line. 4 Reference compounds (set2): Propranolol, Ranitidine, Colchicine and Labetalol.

TC7 cells were seeded at 1×10⁵ cells/cm² on porous polycarbonatemembrane in 96-well Multiscreen™ plates (Millipore). Cells were fedevery 2 to 3 days and the day before the permeability assay.Permeability assays are performed with the cells at days 13-25post-seeding. The assay directions included apical to basolateral (A-B)and basolateral to apical (B-A). The working solution for COTI-2 wasprepared at 10 μM in HBSS-MES (5 mM), pH 6.5, or HBSS-HEPES (5 mM), pH7.4, from a 1 mM DMSO stock solution. The working solution was thencentrifuged and the supernatant was added to the apical or basolateralside with a final DMSO concentration of 1%. The assay was incubated for60 or 40 min with gentle shaking at 37° C. The sampling scheme consistedof donor time zero, donor time 60 or 40 min, and receiver time 60 or 40min. Samples were analyzed via (RP)HPLC-MS/MS using selected reactionmonitoring (SRM). Reference compounds included, propranolol (highlypermeable), labetalol (moderately permeable), ranitidine (poorlypermeable), and colchicine (P-glycoprotein substrate) were included ineach assay. The cell monolayer integrity was confirmed by fluoresceinpermeability assessment (in the A-B direction at pH 7.4 on both sideswith 1 h incubation) after the permeability assay with COTI-2. The cellmonolayer that has a fluorescein permeability of less than 0.5×10⁻⁶ cm/swas considered intact. The apparent permeability coefficient (P_(app))of COTI-2 in the apical to the basolateral and apical direction and thepercent recovery of the compounds were calculated.

For the P-glycoprotein inhibition assay using ³H-digoxin as substrate,the TC7 cells were grown as above.

Two working solutions, working solution I and II, were prepared and eachadded to the apical and basolateral sides, respectively. Workingsolution I consisted of COTI-2 at various concentrations ranging from0.01 to 100 μM in HBSS-HEPES (5 mM)/0.1% BSA, at pH 7.4 from a 10 mMDMSO stock solution. Digoxin (10 μM) and Fluorescein (10 μM) wereincluded in this working solution. The working solution I was then mixedand added to the apical side with a final DMSO concentration of no morethan 1%. Working solution II consisted of various concentrations (0.01to 100 μM) of COTI-2 prepared in HBSS-HEPES (5 mM)/0.1% BSA, at pH 7.4from a 10 mM DMSO stock solution. Digoxin (10 μM) and ³H-digoxin wereincluded in the working solution. The working solution II was then mixedand added to the basolateral side with a final DMSO concentration of nomore than 1%. The assay was incubated for 3 h with gentle shaking at 37°C. The assay was sampled at 3 h and analyzed by liquid scintillationcounting for ³H-digoxin and fluorescence detection for Fluorescein. As areference, verapamil (0.001-0.002 μM) was added to both the A and Bsides. Cell monolayer integrated was assessed by the addition of 10 μMto the apical side and background fluorescence determined from thebasolateral side at time 0 and compared to time 3 h. An apparentpermeability of <0.5×10⁻⁶ cm/s for fluorescein corresponds to an intactmonolayer. The percent inhibition of the permeation of ³H-digoxin wascalculated following scintillation count.

Metabolic Stability:

The experimental conditions are outlined below.

Detected Analytical Assay Substrate/Cofactor Incubation Component MethodMetabolic Test compound (1 μM), 0 and 60 min. Product ion HPLC-Stability (liver NADP (1 mM), 37° C. corresponding MS/MS microsomes, G6P(5 mM), Phosphate to the test human) G6PDHase (1 U/mL) buffer, pHcoumpound via with 0.6% methanol, 7.4 SRM 0.6% acetonitrile (n = 2)Metabolic Test compound (1 μM), 0 and 60 min. Product ion HPLC-Stability (liver NADP (1 mM), 37° C. corresponding MS/MS microsomes, G6P(5 mM), Phosphate to the test monkey, G6PDHase (1 U/mL) buffer, pHcoumpound via Cynomolgus) with 0.6% methanol, 7.4 SRM 0.6% acetonitrile(n = 2) Metabolic Test compound (1 μM), 0 and 60 min. Product ion HPLC-Stability (liver NADP (1 mM), 37° C. corresponding MS/MS microsomes, G6P(5 mM), Phosphate to the test dog, Beagle) G6PDHase (1 U/mL) buffer, pHcoumpound via with 0.6% methanol, 7.4 SRM 0.6% acetonitrile (n = 2)Metabolic Test compound (1 μM), 0 and 60 min. Product ion HPLC-Stability (liver NADP (1 mM), 37° C. corresponding MS/MS microsomes, G6P(5 mM), Phosphate to the test rat, Sprague- G6PDHase (1 U/mL) buffer, pHcoumpound via Dawley) with 0.6% methanol, 7.4 SRM 0.6% acetonitrile (n =2) Abbreviations: CYP: Cytochrome P450 G6P: D-Glucose-6-phosphate, fromSigma, catalog number G-7772 G6PDHase: Glucose-6-phosphatedehydrogenase, from Sigma, catalog number G-4134 HPLC-MS/MS: HPLCcoupled with tandem mass spectrometry (Instrumentation:Thermo Finnigan)HPLC: High performance liquid chromatrography NADP: β-Nicotinamideadenine dinucleotide phosphate, from Sigma, catalog number N-0505 SRM:Selected reaction monitoring

Metabolic stability was determined in human (mixed gender and pool of50) monkey (male Cynomolgus, pool of 6 or more), dog male, beagle, poolof 4 or more) and rat (male Sprague-Dawley, pool of 100 or more) livermicrosomes. Pooled liver microsomes were pre-incubated withNADPH-generating system (1 mM NADP, 5 mM G6P, and 1 U/ml G6PDHase) inphosphate buffer (pH 7.4) containing 3 mM MgCl₂ and 1 mM EDTA in a 2ml-block 96-well plate for 10 min in a 37° C. shaking water bath. Thereaction was initiated by adding COTI-2 (1 μM final concentration) andincubated in a final volume of 400 μl for 0 min and 60 min in the 37° C.shaking water bath. The reaction was stopped by transferring 100 μL ofthe incubation mixture to 100 μl of acetonitrile/methanol (50/50, v/v)in a 0.8 ml V-bottom 96-well plate. Samples were then mixed on a plateshaker for 5 min and centrifuged at 2550×g for 15 min at roomtemperature. Each supernatant (150 μl) was transferred to a cleancluster tube, followed by HPLC-MS/MS analysis on a Thermo Electrontriple-quadrupole system. Four reference substrates (1 μM) were testedsimultaneously with COTI-2. Propranolol and imipramine are relativelystable, whereas verapamil and terfenadine are relatively unstable withhuman liver microsomes. Peak areas corresponding to COTI-2 were recordedby HPLC-MS/MS. Metabolic stability, expressed as percent of COTI-2remaining, was calculated by comparing the peak area of COTI-2 at 60 minto time zero.

CYP450 Inhibition:

The general procedures described previously (Drug Metab Dispos 29:2?3-29) are outlined below:

Detected Analytical Assay Substrate/Cofactor Incubation Component MethodCYP1A Inhibition Test Compound 15 min, Acetaminophen HPLC- (HLM,phenacetin (10 μM), 37° C. MS/MS substrate) Phenacetin (10 μM), NADP(1.3 mM), G6P (3.3 mM), G6PDHase (0.4 U/mL) (n = 2) CYP2B6 InhibitionTest Compound 15 min, Hydroxy- HPLC- (HLM, bupropion (10 μM), 37° C.bupropion MS/MS substrate) Bupropion (100 μM), NADP (1.3 mM), G6P (3.3mM), G6PDHase (0.4 U/mL) (n = 2) CYP2C8 Test Compound 15 min, 6αHydroxypaclitaxel HPLC- Inhibition (HLM, (10 μM), 37° C. MS/MSpaclitaxel Paclitaxel (10 μM), substrate) NADP (1.3 mM), G6P (3.3 mM),G6PDHase (0.4 U/mL) (n = 2) CYP2C9 Test Compound 15 min,4′-hydroxydiclofenac HPLC- Inhibition (HLM, (10 μM), 37° C. MS/MSdiclofenac Diclofenac (10 μM), substrate) NADP (1.3 mM), G6P (3.3 mM),G6PDHase (0.4 U/mL) (n = 2) CYP2C19 Test Compound 15 min,5-hydroxyomeprazole HPLC- Inhibition (HLM, (10 μM), 37° C. MS/MSomeprazole Omeprazole (0.5 μM), substrate) NADP (1.3 mM), G6P (3.3 mM),G6PDHase (0.4 U/mL) (n = 2) CYP2D6 Test Compound 15 min, DextrorphanHPLC- Inhibition (HLM, (10 μM), 37° C. MS/MS dextromethorphanDextromethorphan substrate) (5 μM), NADP (1.3 mM), G6P (3.3 mM),G6PDHase (0.4 U/mL) (n = 2) CYP2E1 Inhibition Test Compound 15 min,6-hydroxychlorzoxazone HPLC- (HLM, (10 μM), 37° C. MS/MS chlorzoxanzoneChlorzoxazone substrate) (100 μM), NADP (1.3 mM), G6P (3.3 mM), G6PDHase(0.4 U/mL) (n = 2) CYP3A Inhibition Test Compound 15 min, 1-hydroxy-HPLC- (HLM, midazolam (10 μM), 37° C. midazolam MS/MS substrate)Midazolam (5 μM), NADP (1.3 mM), G6P (3.3 mM), G6PDHase (0.4 U/mL) (n =2) CYP3A Inhibition Test Compound 15 min, 6β-hydroxytestosterone HPLC-(HLM, (10 μM), 37° C. MS/MS testosterone Testosterone (50 μM),substrate) NADP (1.3 mM), G6P (3.3 mM), G6PDHase (0.4 U/mL) (n = 2)

COTI-2 (10 μM), reference inhibitor, or the vehicle control waspre-incubated with the CYP specific substrate (10 μM) andNADPH-generating system (1.3 mM NADP, 3.3 mM G6P, and 0.4 U/ml G6PDHase)in phosphate buffer (pH 7.4) in a 2 ml-block 96-well plate for 10 min ina 37° C. shaking water bath. The reaction was initiated by adding pooledhuman liver microsomes (mixed gender, pool of 50 donors, 0.2 mg/ml). Thefinal incubation volume was 200 μl. The reaction was allowed for 15 minin the 37° C. shaking water bath and stopped by transferring 100 μl ofthe reaction mixture to 100 μl of acetonitrile/methanol (1/1, v/v) in a0.8 ml V-bottom 96-well plate. Samples are mixed on a plate shaker for 5min and centrifuged at 2550×g for 15 min at room temperature. Thesupernatant (150 μl) is then transferred to a clean cluster tube,followed by HPLC-MS/MS analysis on a Thermo Electron triple-quadrupolesystem to detect the metabolite. Each CYP specific inhibitor was testedsimultaneously with reference compounds at several concentrations toobtain the IC₅₀ value. Peak areas corresponding to metabolite wererecorded. The percent of control activity was calculated by comparingthe peak area in incubations containing COTI-2 at 15 min to the controlsamples containing the same solvent vehicle at 15 min. Subsequently, thepercent inhibition was calculated by subtracting the percent controlactivity from 100.

Results and Discussion:

Plasma Protein Binding:

The binding of pharmaceutical agents to plasma proteins, mostly to serumalbumin, lipoprotein and α-acid glycoprotein, is one of many factorsthat influences the pharmacokinetic and pharmacodynamic properties of anagent. It is widely accepted that a compound's efficacy may be affectedby the degree to which it binds to plasma proteins, such that theunbound concentration of an agent in plasma is available to diffuseextra-vascular spaces (organs and tissues), cell membranes, and interactwith the pharmacological target (Clin Pharmacokinet 39: 345-367). Theplasma-bound form of the therapeutic agent can also serve as a reservoirfrom which the agent is released to an unbound form. Consequently, testshave been designed to determine the bound & unbound fractions of thetotal compound concentration. The two most commonly employed methodsused to determine plasma protein-binding of compounds areultrafiltration and equilibrium dialysis (Clin Pharmacokinet 23: 449-468& Fundam Clin Pharmacol 4(suppl2): 151 s-161 s).

COTI-2 was evaluated for binding plasma proteins in 3 species, namelyhuman, monkey, and dog, using an equilibrium dialysis technique thatseparates the fraction of compound that is unbound from that which isbound. As a control, a calibration sample was utilized to determinepercent recovery. Both percent protein binding and recovery werecalculated (Table 26) and compared against plasma protein binding of thereference compounds, acetbutolol, quinidine and warfarin, whichrepresent compounds with low, medium, and high binding to plasmaproteins, respectively (Table 27). The data indicate that COTI-2exhibits high binding to plasma proteins from all 3 species tested(>99%). These data, however, should be interpreted with caution sincethe percent recovery values are low (<50%). The percent recovery is anindicator of the reliability of the percent protein bound values. Lowrecovery values indicate that the test compound is lost during the assaydue to non-specific interactions (e.g., binding to the apparatus,membrane, etc) or due to degradation by enzymatic activity. Having saidthat, these data indicate that since the plasma protein binding ofCOTI-2 among the 3 species examined is highly similar, plasma proteinlevels are not anticipated to complicate the understanding ofinterspecies pharmacodynamic and toxicological effects. This isparticularly important when evaluating the nonclinical toxicologicaldata generated in rats and dogs necessary for First-in-Human (FIH)trials.

TABLE 26 Percent protein bound and recovery of COTI-2 in human, rat, anddog plasma. Assay Cerep Client Test Compound Compound Concentration %Protein Bound % Recovery I.D. I.D. (M) 1^(st) 2^(nd) Mean 1^(st) 2^(nd)Mean Plasma Protein Binding (human) 15731-1 COTI2- 1.0E−05 99.8 >99.999.8 6.7 2.9 4.8 M05 Plasma Protein Binding (rat, mixed breeds) 15731-1COTI2- 1.0E−05 >99.9 >99.9 >99.9 16.4 25.1 20.7 M05 Plasma ProteinBinding (dog, mixed breeds) 15731-1 COTI2- 1.0E−05 99.1 99.7 99.4 42.632.8 37.7 M05

TABLE 27 Percent protein bound of reference compounds, acebutolol,quinidine, and warfarin, in human, rat, and dog plasma. Test AssayConcentration % Protein Bound Reference Compound (M) 1^(st) 2^(nd) MeanPlasma Protein Binding (human) Acebutolol 1.0E−05 14.9 12.8 13.8Quinidine 1.0E−05 52.5 48.1 50.3 Warfarin 1.0E−05 94.2 96.4 95.3 PlasmaProtein Binding (rat, mixed breeds) Acebutolol 1.0E−05 8.0 3.4 5.7Quinidine 1.0E−05 67.7 62.8 65.2 Warfarin 1.0E−05 97.8 97.6 97.7 PlasmaProtein Binding (dog, mixed breeds) Acebutolol 1.0E−05 18.9 5.3 12.1Quinidine 1.0E−05 70.8 83.4 77.1 Warfarin 1.0E−05 95.8 96.1 95.9In Vitro Absorption (Pgp Substrate or Inhibitor):

An understanding of intestinal transport is crucial for evaluating thepotential for oral dosing of pharmacological agents (Drug Discov Today13: 379-393). As a rule, a given pharmacological agent will interactwith membrane transporter(s) at some point of its route in the body. Ofparticular importance are the multiple drug resistance (MDR)transporters, especially P-glycoprotein (Pgp), which plays an importantrole in limiting oral drug delivery by active efflux of pharmacologicalagents from the intestinal mucosa and into the lumen (Xenobiotica 38:802-832). Pgp is also located at the blood-brain barrier among othertissues, thus may exclude pharmacological agents targeted to the centralnervous system from the brain affects and result in poor brainpenetration. In addition, Pgp confers multidrug-resistance to cancercells (Nat Rev Drug Discov 5: 219-234). The bi-directional transportassay, which also measures intestinal permeability, is regarded as thedefinitive assay for identifying Pgp substrates and inhibitors becauseit measures drug efflux in a more direct manner than other methods(Xenobiotica 38: 802-832).

In vitro cell culture systems, such as Caco-2 and TC-7, have been usedto predict human in vivo intestinal permeability (Cell Biol Toxicol 21:1-26 & Pharm Res 15: 726-733). The Caco-2 cell line, derived fromgastrointestinal tumors, has been used for decades to study intestinalpermeability. The TC-7 cell line, which is a sub-clone of Caco-2, hasbeen shown to be a valuable alternative to the Caco-2 cells based onmorphological, biochemical, and drug transport characteristics.Furthermore, the atypically-located Pgp, the monocarboxylic acidtransporter, the dipeptided transporter, and the transporter for largeneutral amino acids are expressed in both Caco-2 and the subclone TC-7cells at similar levels. Therefore, the latter cell line was used forevaluating the intestinal permeability of COTI-2 and for determiningwhether COTI-2 is a Pgp substrate or inhibitor.

The TC-7 cells were grown to a monolayer and 10 μM COTI-2 added eitherto the apical (A) or basolateral (B) side of the monolayer to measurepermeability. Verapamil, which is an inhibitor of Pgp, was added on boththe apical and basolateral sides of the monolayer in the presence ofCOTI-2, as a control to determine whether the efflux of COTI-2 isPgp-mediated. The data are represented as apparent permeabilitycoefficient (P_(app [)10⁻⁶ cm/s]) and percent recovery. The referencecompounds included in the assay are propranolol (highly permeable),labetalol (moderately permeable, ranitidine (poorly permeable), andcolchicines (P_(app) substrate) (Table 28). The TC-7 permeabilityclassification is as follows:

P_(app)<2×10⁻⁶ cm/s Low permeability

2×10⁻⁶ cm/s<P_(app)<20×10⁻⁶ cm/s Medium permeability

P_(app)>20×10⁻⁶ cm/s High permeability

Although the A-B permeability of COTI-2 was less than 0.3×10⁻⁶ cm/s(Table 29), which indicates low permeability according to the TC-7permeability classification, the mean recovery (22%) suggests the lossof COTI-2 during the assay due to non-specific interactions ordegradation. In fact, pharmacokinetics data in rats following a singleoral dose of 20 mg/kg of the parent COTI-2 form demonstrated 17.7%bioavailability by comparison against IV administration. Therefore,based on this previous in vivo pharmacokinetic study, the unformulatedand un-ground parent base COTI-2 does appear to penetrate the intestinalbarrier.

To evaluate whether a compound is a Pgp substrate, it is important tocalculate the efflux ratio (E ratio). The E ratio is the permeabilityvalue in the B-A direction divided by the permeability value in the A-Bdirection. Compounds with an E ratio greater than 2 are possible Pgpsubstrates. Furthermore, in the presence of verapamil, the net effluxratio should approach unity or be significantly decreased from the Eratio in the absence of verapamil to indicate a Pgp substrate. The Eratio of COTI-2 in the absence of verapamil is approximately 4.5,suggesting that COTI-2 is a possible substrate of Pgp (Table 29).However, in the presence of verapamil, the E ratio is about 4.0, whichis not significantly different than without verapamil (4.5). Thus,COTI-2 is unlikely to be a Pgp substrate. There may be some effluxoccurring, but it is not related to Pgp. Verapamil may be affectingother efflux systems in the cells. If COTI-2 were a Pgp substrate, onewould expect not only the ratio to be reduced, but also the B-Apermeability to be decreased in the presence of verapamil sinceverapamil decreases B-A permeability of a Pgp substrate by inhibitingPgp. In fact, the B-A permeability of COTI-2 increased in the presenceof verapamil and the B-A permeability of COTI-2 in the absence ofverapamil (Table 29) is lower than all the reference compounds (Table28). These data allow for a confident assessment that COTI-2 is not asubstrate of Pgp.

TABLE 28 The permeability and percent recovery of the referencecompounds colchicines, labetalol, propranolol, and ranitidine determinedusing TC-7 cell lines. Assay Test Permeability Percent RecoveryReference Concentration 1^(st) 2^(nd) Mean 1^(st) 2^(nd) Mean Compound(M) (10⁻⁶ cm/sec) (10⁻⁶ cm/sec) (10⁻⁶ cm/sec) (%) (%) (%) A-BPermeability (TC7, pH 7.4/7.4) Colchicine 1.0E−05 0.04 0.02 0.0 98 98 98Labetalol 1.0E−05 7.93 5.69 6.8 80 79 80 Propranolol 1.0E−05 54.87 48.5951.7 58 59 58 Ranitidine 1.0E−05 0.48 0.48 0.5 98 101 99 B-APermeability (TC7, pH 7.4/7.4) Colchicine 1.0E−05 6.43 6.45 6.4 103 100101 Labetalol 1.0E−05 39.56 37.42 38.5 103 97 100 Propranolol 1.0E−0518.70 18.81 18.8 96 96 96 Ranitidine 1.0E−05 4.20 4.20 4.2 110 110 110

TABLE 29 The permeability and percent recovery of COTI-2 determinedusing TC-7 cell lines. Assay Percent Cerep Client Test PermeabilityRecovery Compound Compound Concentration 1^(st) 2^(nd) Mean 1^(st)2^(nd) Mean I.D. I.D. (M) (10⁻⁶ cm/sec) (10⁻⁶ cm/sec) (10⁻⁶ cm/sec)Flags (%) (%) (%) A-B Permeability (TC7, pH 7.4/7.4) 15731-1 COTI2-1.0E−05 0.33 0.32 <0.3 BLQ 23 21 22 M05 A-B Permeability (TC7, pH7.4/7.4) + verapamil 15731-1 COTI2- 1.0E−05 2.37 3.96 3.2 17 13 15 M05B-A Permeability (TC7, pH 7.4/7.4) 15731-1 COTI2- 1.0E−05 1.37 1.47 1.4114 90 102 M05 B-A Permeability (TC7, pH 7.4/7.4) + verapamil 15731-1COTI2- 1.0E−05 15.85 9.13 12.5 49 46 47 M05 BLQ Below the Limit ofQuantitation. Test compound was well detected in donor samples but notdetected in receiver samples. The concentration of test compound inreceiver sample was below the limit of quantitation.

A compound can either be a substrate or inhibitor of Pgp. In order todetermine whether COTI-2 is an inhibitor of Pgp an inhibition assay wasperformed using Digoxin, which is a well-known substrate of Pgp. TheIC₅₀ of COTI-2 was determined by determining amount of radiolabelleddigoxin in the presence or absence of various concentrations of COTI-2.Verapamil was used as a positive control. The IC₅₀ of COTI-2 (39 μM) wasless than 10-fold the IC₅₀ of verapamil (5.5 μM), which is a stronginhibitor of Pgp (Table 30). These data indicate that COTI-2 is likelyto be a moderate inhibitor of Pgp. The role of Pgp in cancer therapy isbecoming increasingly recognized, since Pgp efflux has been linked tomultidrug resistance (IDrugs 5: 349-355 & Nat Rev Drug Discov 5:219-234). In fact, co-administration of Pgp inhibiting agents as part ofchemotherapy is an area that is being extensively studied in the hope offinding an ideal drug that inhibits Pgp but does not cause wide-rangingadverse effects (Xenobiotica 38: 802-832). Therefore, an anticancertherapeutic like COTI-2, which inhibits Pgp efflux can potentiallyincrease oral bioavailability or reduce multidrug resistance.

TABLE 30 The Pgp inhibition of COTI-2 and verapamil in the TC-7 cellline. Assay IC₅₀ Cerep Compound I.D. Client Compound I.D. (M) n_(H)P-glycoprotein Inhibition (TC7, ³H-digoxin substrate) 15731-1 COTI2-M053.9E−05 0.5 Assay IC₅₀ Reference Compound (M) n_(H) P-glycoproteinInhibition (TC7, ³H-digoxin substrate) Verapamil 5.5E−06 0.9Metabolic Stability:

The clearance and bioavailability of most pharmacological agents arestrongly influence by their first-pass metabolism in the liver (Nat RevCancer 6: 546-558). It is possible to estimate the relative hepatic“metabolic stability” in vitro by incubating compounds with livermicrosomes and determining the initial versus the final amount of thetest compound in the incubation mixtures.

The relative stability of 1.0 μM COTI-2 in liver microsomes of human,monkey, dog, and rat was evaluated at 60 min post-incubation and datapresented as mean percent remaining of COTI-2. Four reference substrates(1.0 μM) were tested, including propranolol and imipramine, which arerelatively stable, whereas verapamil and terfenadine, which arerelatively unstable with human liver microsomes (Table 32). COTI-2appears to be most stable in human liver microsomes compared to monkey,dog, and rat liver microsomes (Table 31). These data, which will be usedto guide preclinical toxicological studies, predict that COTI-2 will becleared at a higher rate in the preclinical in vivo models relative toclearance in humans. Furthermore, the stability of COTI-2 in human livermicrosomes (31% remaining at 60 min) appears to be moderate compared tothe reference substrates.

TABLE 31 Metabolic stability of COTI-2 in human, monkey, dog, and ratliver microsomes as indicated by percent remaining. Mean Test ParentAssay Client Concentration Remaining Cerep Compound I.D. Compound I.D.(M) (%) Metabolic Stability (liver microsomes, human) 15731-1 COTI2-M051.0E−06 31 Metabolic Stability (liver microsomes, monkey, Cynomolgus)15731-1 COTI2-M05 1.0E−06 2 Metabolic Stability (liver microsomes, dog,Beagle) 15731-1 COTI2-M05 1.0E−06 5 Metabolic Stability (livermicrosomes, rat, Sprague-Dawley) 15731-1 COTI2-M05 1.0E−06 1

TABLE 32 Metabolic stability of reference compounds in human, monkey,dog and rat liver microsomes as indicated by percent remaining. TestParent Remaining Assay Concentration 1^(st) 2^(nd) Mean ReferenceCompound (M) (%) (%) (%) Metabolic Stability (liver microsomes, human)Imipramine 1.0E−06 68.8 71.1 70 Propranolol 1.0E−06 77.6 76.7 77Terfenadine 1.0E−06 9.2 8.7 9 Verapamil 1.0E−06 17.3 17.0 17 MetabolicStability (liver microsomes, monkey, Cynomolgus) Imipramine 1.0E−06 0.30.3 0 Propranolol 1.0E−06 23.3 21.5 22 Terfenadine 1.0E−06 0.6 0.5 1Verapamil 1.0E−06 0.4 0.1 0 Metabolic Stability (liver microsomes, dog,Beagle) Imipramine 1.0E−06 1.2 0.9 1 Propranolol 1.0E−06 24.4 25.4 25Terfenadine 1.0E−06 88.2 88.9 89 Verapamil 1.0E−06 18.1 18.0 18Metabolic Stability (liver microsomes, rat, Sprague-Dawley) Imipramine1.0E−06 0.3 0.2 0 Propranolol 1.0E−06 0.0 0.0 0 Terfenadine 1.0E−06 4.13.5 4 Verapamil 1.0E−06 13.3 13.5 13CYP450 Inhibition:

The metabolism-based drug-drug interactions occur when a pharmacologicalagent inhibits or induces the activity of a drug metabolizing enzyme,which catalyzes the metabolism of any concomitant drugs. Themetabolism-based drug-drug interaction is one of the major factors thatcause drug failures during drug development. The major metabolicpathways involved can be classified into 2 groups, phase I and phase IIreactions (J Clin Pharmacol 41: 1149-1179). The cytochrome P450(CYP450), which is an enzyme superfamily of hemoproteins that catalyzethe phase I reactions, are estimated to account for thebiotransformation of approximately 60% of the commonly prescribed drugsin the US. A number of in vitro assays for CYP450 inhibition have beendeveloped, which differ in CYP enzyme source and composition (i.e.,recombinant cDNA expressed human CYP isozymes, human liver microsomes,probe substrates, and detection [e.g., LC-MS, fluorescence,radioactivity]). Microsomes and recombinant P450 enzymes are thepreferred test system as they are more readily available than humanhepatocytes, and P450 kinetic measurements are not confounded with othermetabolic processes or cellular uptake (Drug Metab Dispos 31: 815-832).

The metabolism of COTI-2 by the CYP enzymes of major importance in drugmetabolism was analyzed by determining the percent inhibition relativeto solvent only control (which is considered 100% uninhibited enzymeactivity). Inhibition of greater than 50% suggests an inhibitoryactivity of a test compound towards a specific CYP450 enzyme andrequires further investigation by IC₅₀ determination. The inhibition byCOTI-2 for all the CYP450 enzymes tested was well below 50%, thusindicating that it is not likely to be a significant inhibitor of theCYP enzymes (Table 33). The negative inhibition values, resulting frominteraction of the components in the reaction mixture, also indicatethat COTI-2 is not an inhibitor of CYP enzymes. Represented in Table 34are the IC₅₀ values of known inhibitors of each of the assayed CYP450enzymes.

TABLE 33 Percent inhibition of CYP450 enzymes by COTI-2 relative tocontrol (vehicle alone) values as determined by a human liver microsomalassay. Test % Inhibition Assay Cerep Client Concentration of ControlCompound I.D. Compound I.D. (M) Values CYP1A Inhibition (HLM, phenacetinsubstrate) 15731-1 COTI2-M05 1.0E−05 −2 CYP2B6 Inhibition (HLM,bupropion substrate) 15731-1 COTI2-M05 1.0E−05 5 CYP2C8 Inhibition (HLM,paclitaxel substrate) 15731-1 COTI2-M05 1.0E−05 10 CYP2C9 Inhibition(HLM, diclofenac substrate) 15731-1 COTI2-M05 1.0E−05 2 CYP2C19Inhibition (HLM, omeprazole substrate) 15731-1 COTI2-M05 1.0E−05 4CYP2D6 Inhibition (HLM, dextromethorphan substrate) 15731-1 COTI2-M051.0E−05 2 CYP2E1 Inhibition (HLM, chlorzoxazone substrate) 15731-1COTI2-M05 1.0E−05 −2 CYP3A Inhibition (HLM, midazolam substrate) 15731-1COTI2-M05 1.0E−05 7 CYP3A Inhibition (HLM, testosterone substrate)15731-1 COTI2-M05 1.0E−05 −4

TABLE 34 IC₅₀ values of known inhibitors of the CYP450 enzymes asdetermined by a human liver microsomal assay. Assay IC50 ReferenceCompound (M) n_(H) CYP1A Inhibition (HLM, phenacetin substrate)Furafylline 4.9E−06 0.8 CYP2B6 Inhibition (HLM, bupopion substrate)Clopidogrel 6.4E−07 2.7 CYP2C8 Inhibition (HLM, paclitaxel substrate)Nicardipine 2.3E−06 1.2 CYP2C9 Inhibition (HLM, diclofenac substrate)Sulfaphenazole 9.5E−07 1.1 CYP2C19 Inhibition (HLM, omeprazolesubstrate) Oxybutynin 6.6E−06 1.1 CYP2D6 Inhibition (HLM,dextromethorphan substrate) Quinidine 1.4E−07 0.9 CYP2E1 Inhibition(HLM, chlorzoxazone substrate) 4-Mehylpyrazole 3.3E−07 1.0 CYP3AInhibition (HLM, midazolam substrate) Ketoconazole 2.4E−07 1.8 CYP3AInhibition (HLM, testosterone substrate) Ketoconazole 5.0E−07 0.9

Example 24 In Vivo Pharmacokinetics

Pharmacokinetic studies in animal models provide valuable informationthat enables prediction of metabolism in humans. There are a largevariety of animal models that allow for the pharmacokinetic evaluationof compounds by providing information on serum, plasma, and tissuelevels. One of the major differences between animal and humanpharmacokinetics is that a compound is cleared at a faster rate inanimals, particularly rodents, compared to clearance in humans (Int JAntimicrob Agents 19: 261-268). As a result, it is important to keepthis phenomenon in mind during extrapolations from animal to humanmetabolism. An initial pharmacokinetic assessment of COTI-2,administered both intravenously and orally, in its parent formulationand as both an oxalate and tartrate salt were evaluated in rats.

Methods:

Intrinsic Clearance and Half-Life Determination:

The incubation mixture (500 μl) consisted of 100 μl of COTI-2, 150 μl ofliver microsomal fractions (1.67 mg/ml rat/human) 35.5 μl of 100 mMphosphate buffer and 214.5 μl co-factor (NADPH, UDPGA, and MgCl₂). TheCOTI-2 compound mixture and co-factor were pre-warmed at 37° C. for 5min, with shaking at 150 rpm. The reaction was started by addingco-factor to the test compound mixture. The final concentration of theincubation mixture was as follows:

Microsomal protein: 0.5 mg/ml NADPH: 0.823 mM UDPGA: 5 mM MgCl₂: 1 mM

Blank incubation was performed with the co-factors. The sampling timepoints included 0 and 240 min for the blank sample, and 0, 20, 40, 60,120, 180, and 240 min for COTI-2. At each time point 50 μl of sample wasadded to 150 μl of methanol to stop the reaction. The sample plates werecentrifuged at 4200 rpm for 15 min and the supernatant was injected intoLC-MS/MS for analysis. The percent compound remaining, rate ofelimination (K_(el)), half-life (T_(1/2)), and intrinsic clearance (CL)using standard calculations.

Results and Discussion:

A single oral or IV dose of the COTI-2 free-base or the aforementionedsalt forms was administered to groups of male and female rats and bloodsamples collected and processed at several time-points post-treatment.The pharmacokinetic parameters following IV administration aresummarized in Table 35. The extrapolated C_(initial) values for COTI-2oxalate and tartrate formulations were approximately 4-fold and 2-foldhigher than the parent formulation, respectively. The calculated extentof exposure over the 24-hour post-dose sampling period (AUC_(0-24 hrs))for the oxalate and tartrate formulations were approximately 2-foldhigher than the parent COTI-2. The rate of elimination (K_(e)) of COTI-2oxalate was approximately 2-fold higher than parent and tartrateformulations, corresponding to a decreased half-life of elimination(T_(1/2(e))), total half-life (T_(1/2(TOTAL))) and mean residence time(MRT) in plasma. The parent COTI-2 exhibited the highest rate ofclearance (CL) from plasma, approximately 1.4- and 2.3-fold higher thanthe oxalate and tartrate formulations, respectively, due to an increasedcalculated volume of distribution (V_(d)).

TABLE 35 A summary of the pharmacokinetic parameters following IVdosing. Parent Oxalate Tartrate COTI2 COTI2 COTI2** Parameter Unit (5mg/kg) (5 mg/kg) (5 mg/kg) C_(initial) ng/mL 1310.0 5205.7 2016.2 K_(e)hr 0.023 0.052 0.021 T_(1/2(e)) hr 30.28 13.39 32.58 AUC_(0-24 hrs) ng *hr/mL 9184.3 20901.1 20184.0 AUC_(0-∞) ng * hr/mL 19958.3 28614.246650.4 AUMC_(0-∞) ng * hr²/mL 813242.7 501668.9 2069699.4 MRT hr 40.717.5 44.4 V_(d) l 23.79 4.62 11.65 CL mL/hr/kg 250.52 174.74 107.18T_(1/2 (TOTAL))* hr 30.3 13.4 32.6 *calculated from V_(d) and CL **n = 5for 24-hour time point: data from female rat #075 omitted

The pharmacokinetic parameters following oral dosing are summarized inTable 36. The C_(max) values for COTI-2 oxalate and tartrateformulations were approximately 1.75-fold and 3.3-fold higher than theparent formulation, respectively, and the T_(max) values weresignificantly shorter (15 min vs. 4 h). The calculated rate ofabsorption (K_(a)) of parent COTI-2 was approximately 3-fold higher thanthe other formulations; this was due to increasing plasma concentrationsof parent COTI-2 at the 15- and 30-min post-dose time points, comparedto decreasing concentration of COTI-2 oxalate and tartrate at the 30-minpost-dose time point. The calculated extent of exposure over the 24-hourpost-dose sampling period (AUC_(0-24 hrs)) for the oxalate and tartrateformulations were comparable to parent COTI-2, with extent of exposureto the tartrate formulation slightly increased by approximately1.4-fold. The rates (K_(e)) and times (T_(1/2(e))) of elimination andmean residence times (MRT) of all formulations were comparable. Theparent COTI-2 exhibited the highest rate of clearance (CL) from plasma,approximately 2-fold higher than the oxalate and tartrate formulations.The bioavailability (F) of parent COTI-2, COTI-2 oxalate and COTI-2tartrate formulations after oral administration, as determined bycomparison with IV administration, was 17.7%, 7.6% and 11.0%,respectively.

TABLE 36 A summary of the pharmacokinetic parameters following oraldosing. Parent Tartrate COTI2 Oxalate COTI2 COTI2 Parameter Unit (20mg/kg) (20 mg/kg) (20 mg/kg) C_(max) ng/ml 882.0 1541.7 2903.8 T_(max)hr 4.0 0.25 0.25 K_(a) hr⁻¹ 0.681 0.210 0.230 T_(1/2(a)) hr 1.02 3.303.02 K_(e) hr⁻¹ 0.055 0.070 0.078 T_(1/2(e)) hr 12.49 9.97 8.86AUC_(0-24 hrs) ng * hr/mL 6500.6 6314.8 8893.5 AUC_(0-∞) ng * hr/mL7578.6 7237.8 10053.8 AUMC_(0-∞) ng * hr²/mL 81677.0 72613.6 96208.9 MRThr 10.8 10.0 9.6 CL* mL/hr/kg 467.10 210.01 200.92 F % 17.7 7.6 11.0*calculated from F as determined by comparison with IV dose kinetics

The graphical representation of the plasma concentration of each agentfollowing IV and oral dosing are represented in FIGS. 52A-B.

Example 25 Determination of Plasma Exposure and Efficacy of COTI-2 InVivo as a Single Agent and in Combination with Taxol® (Paclitaxel) inthe AN3-CA Human Endometrial Tumor Xenograft Model

The purpose of this study was to evaluate the antitumor activity ofCOTI-2 as a single agent and in combination with paclitaxel in theAN3-CA human endometrial tumor xenograft model.

Materials and Methods:

Reagents and Compounds

COTI-2 mono-HCl salt (Lot#CKL-2-148) was stored at −20° C. until readyfor use. A stock solution of COTI-2 was made at a concentration of 12mg/ml; 235 mg of COTI-2 was mixed with 4.84 g of Captisol®(Lot#CY-04A-05006 Cyclex Pharmaceuticals; Lenexa, Kans.) in 10.0 ml ofSterile Water for Injection (Hospira, Inc., Lake Forest, Ill.). Thesolution was prepared in a 60 ml amber vial and left to stir at 400 rpmfor 1 h. The solution was then filtered with a 0.2 μm PVDF filter toachieve the 12 mg/ml stock concentration. The stock was further dilutedto 6 mg/ml with 50 mM Captisol® solution in sterile water (at pH 3.0).The 6 mg/ml solution was also filtered with a 0.2 μm PVDF filter. Taxol®(paclitaxel) (Lot#U026849AA) was received from Hospira, Inc. (LakeForest, Ill.), and diluted in a 0.9% NaCl solution (Baxter, Deerfield,Ill.) to a concentration of 0.5 mg/ml to deliver 5 mg/kg in a 10 ml/kgdose volume. All preparations were made fresh prior to theiradministration.

Cell Culture

The AN3-CA endometrial tumor cell line was received from American TypeCulture Collection (ATCC, Manassas, Va.). Cultures were maintained inRPMI 1640 (Hyclone, Logan, Utah) supplemented with 5% fetal bovineserum, and housed in a 10% CO₂ atmosphere. The cultures were expanded intissue culture flasks at a 1:7 split ratio until a sufficient amount ofcells were harvested.

Animals

Female athymic nude mice (CrTac:NCR-Foxn1^(nu)) were supplied by Taconic(Germantown, N.Y.). Mice were received at four weeks of age and wereacclimated for seven days prior to handling. The mice were housed inmicroisolator cages (Lab Products, Seaford, Del.) and maintained underspecific pathogen-free conditions. The mice were fed PicoLab® irradiatedmouse chow (Lab Diet, Richmond, Ind.) and autoclaved water was freelyavailable. All procedures were carried out under the institutionalguidelines of TGen Drug Development Services Institutional Animal Careand Use Committee (Protocol #09002, Approved February 2009).

AN3-CA Human Endometrial Tumor Xenograft Model

Eighty-eight mice were inoculated subcutaneously in the right flank with0.1 ml of a 50% RPMI/50% Matrigel™ (BD Biosciences, Bedford, Mass.)mixture containing a suspension of AN3-CA tumor cells (approximately1×10⁷ cells/mouse). Nine days following inoculation, tumors weremeasured using calipers and tumor weight was calculated using the animalstudy management software, Study Director V.1.6.80 (Study Log) (CancerRes 59: 1049-1053). Seventy mice with tumor sizes of 52-243 mg werepair-matched into the seven groups of ten mice by random equilibration,using Study Director (Day 1). Body weights were recorded when the micewere pair-matched. Body weights were taken twice weekly thereafter inconjunction with tumor measurements. On Day 1, COTI-2, vehicle control,and paclitaxel, were administered intravenously. The dosing schedule forCOTI-2 and the vehicle control was three times weekly until study end.The schedule for paclitaxel was daily for five days (QD×5). This dosingregimen was repeated only to the paclitaxel single agent group startingon Day 27. The paclitaxel single agent group was treated with COTI-2 (25mg/kg) for five days (QD×5) starting on Day 32. When the mean tumorweight of each mouse reached an approximate end-point of 2000 mg, micewere sacrificed with regulated CO₂.

Data and Statistical Analysis:

Mean tumor growth inhibition (TGI) was calculated utilizing thefollowing formula:

${TGI} = {\left\lbrack {1 - \frac{\left( {{\overset{\_}{\chi}}_{{Treated}_{({Final})}} - {\overset{\_}{\chi}}_{{Treated}_{({{Day}\; 1})}}} \right)}{\left( {{\overset{\_}{\chi}}_{{Control}_{({Final})}} - {\overset{\_}{\chi}}_{{Control}_{({{Day}\; 1})}}} \right)}} \right\rbrack \times 100\%}$

Tumors that regressed from the Day 1 starting size were removed from thecalculations. Individual tumor shrinkage (TS) was calculated using theformula below for tumors that showed regression relative to Day 1 tumorweight. The mean tumor shrinkage of each group was calculated andreported.

${TS} = {\left\lbrack {1 - \frac{\left( {{Tumor}\mspace{14mu}{Weight}_{({Final})}} \right)}{\left( {{Tumor}\mspace{14mu}{Weight}_{({{Day}\; 1})}} \right)}} \right\rbrack \times 100\%}$

All statistical analyses in the xenograft study were performed withGraphPad Prism® v4 software. Differences in tumor weights were confirmedusing the Analysis of Variance (ANOVA) with the Tukey's Post Test andthe One-tailed Student's T test. Increase in survival fraction wasconfirmed by the log rank test.

Results and Discussion:

This study determined the antitumor effects of COTI-2 when administeredas a single agent and when administered in combination with paclitaxel.The treatment regimens were tested against the AN3-CA human endometrialtumor xenograft model. Efficacy was assessed by comparison of tumorweights (Table 37 and FIG. 53) and survival at an endpoint of 2000 mg(Table 38).

Overall, single agent treatment of the test agent resulted in slighttoxicity in the higher doses and in combination with paclitaxel. Miceexhibited lethargy and hypoactivity with continued dosing at 50 mg/kg.Deaths in the 25 mg/kg and 50 mg/kg groups were possibly due to drugtoxicity and tumor burden, since both groups had tumors over 1500 mg atthe time of death. COTI-2 as a single agent did not show any statisticaldifferences in tumor weights or survival, when compared to control(Tables 37 and 38). However, in combination with paclitaxel, COTI-2showed positive interaction with 90-100% tumor regression in thecombination groups compared to 50% regression observed in the singleagent paclitaxel treated group. The combination treatments significantlyincreased survival relative to paclitaxel alone survival (Table 38). ACOTI-2 dose level dependency was also observed in the combinationtreatments (FIG. 53). In conclusion, COTI-2 dosed at 25 mg/kg incombination with paclitaxel resulted in the highest anti-tumor activityagainst the AN3-CA human endometrial xenograft model.

TABLE 37 Summary of tumor weights between groups on Day 13 of the study.Day 13 Maximum Mean Day weight tumor 13 Day Dose Schedule loss (%)weight TGI 13 Group N (mg/kg) Route (Day) (Day #) (mg) (%) TS¹ CS²Vehicle 10 — IV 3X Weekly  5.6% (Day 9) 1,667.8 ± 240.7 — — 0 Control toend COTI-2 10 12.5 IV 3X Weekly  8.2% (Day 21) 1,828.9 ± 265.8 — — 0 toend COTI-2 10 25.0 IV 3X Weekly  4.7% (Day 6) 2,108.7 ± 263.0 — — 0 toend COTI-2 10 50.0 IV 3X Weekly  5.4% (Day 21) 1,542.7 ± 217.2  7.8 — 0to end Paclitaxel 10 2.0 IV QD × 5  8.0% (Day 6)   175.1 ± 62.44 89.733.9 0 (5/10) COTI-2 10 12.5 IV 3X Weekly 11.0% (Day 6)   58.4 ± 12.798.9 54.5 3 Paclitaxel 2.0 IV to end (9/10) QD × 5 COTI-2 10 25.0 IV 3XWeekly 10.8% (Day 6)  48.0 ± 8.7 — 55.6 4 Paclitaxel 2.0 IV to end(10/10)  QD × 5 TS¹ = Tumor Shrinkage CS² = Complete Shrinkage

TABLE 38 Summary of survival between groups at an end point of 2000 mg.Dose Mean days (mg/ Schedule of Survival Group N kg) Route (Day) (Day ±SEM) Deaths Vehicle 10 — IV 3X Weekly to 17.0 ± 1.0 0 Control end COTI-210 12.5 IV 3X Weekly to 16.7 ± 0.8 0 end COTI-2 10 25.0 IV 3X Weekly to15.8 ± 0.9 1 end COTI-2 10 50.0 IV 3X Weekly to 17.2 ± 0.7 1 endPaclitaxel 10 2.0 IV QD × 5 32.1 ± 1.5 0 COTI-2 10 12.5 IV 3X Weekly to33.9 ± 1.3 0 Paclitaxel 2.0 IV end QD × 5 COTI-2 10 25.0 IV 3X Weekly to35.3 ± 0.9 0 Paclitaxel 2.0 IV end QD × 5

Example 26 Evaluation of COTI-2 as a Single Agent and in Combinationwith Doxil® in the A2780 Human Ovarian Tumor Xenograft Model

The purpose of this study was to determine the single agent efficacy ofCOTI-2 in the A2780 human ovarian tumor xenograft model. COTI-2 was alsostudied in comparison, as well as in combination, with the standardagent Doxil®.

Materials and Methods:

Reagents and Compounds

COTI-2 mono-HCl salt (Lot#CKL-2-148) was stored at −20° C. until readyfor use. A stock solution of COTI-2 mono-HCl salt was made at aconcentration of 12 mg/ml; 235 mg of COTI-2 was mixed with 4.84 g ofCaptisol® (Lot#CY-04A-05006 Cyclex Pharmaceuticals; Lenexa, Kans.) in10.0 ml of Sterile Water for Injection (Hospira, Inc., Lake Forest,Ill.). The solution was prepared in a 60 ml amber vial and left to stirat 400 rpm for 1 h. The solution was then filtered with a 0.2 μm PVDFfilter to achieve the 12 mg/ml stock concentration. The stock wasfurther diluted to 6 mg/ml with 50 mM Captisol® solution in sterilewater (at pH 3.0). The 6 mg/ml solution was also filtered with a 0.2 μmPVDF filter. Doxil® (Lot#0821642), was manufactured by Ortho Biotech(Raritan, N.J.), and was stored at 4° C. until used. It was diluted in a0.9% NaCl solution to a concentration of 0.2 mg/ml to deliver a 2 mg/kgdose intravenously in a 10 ml/kg dose volume. All preparations were madefresh prior to their administration.

Cell Culture

The A2780 human ovarian tumor cell line was received from American TypeCulture Collection (ATCC, Manassas, Va.). Cultures were maintained inRPMI 1640 (Hylcone Labs, Logan, Utah) supplemented with 5% fetal bovineserum, and housed in a 5% CO2 atmosphere. The cultures were expanded intissue culture flasks at a 1:4 split ratio until a sufficient amount ofcells were harvested.

Animals

Female athymic nude mice (CrTac: NCR-Foxn1^(nu)) were supplied byTaconic (Germantown, N.Y.). Mice were received at four weeks of age. Allmice were acclimated for seven days prior to handling. The mice werehoused in microisolator cages (Lab Products, Seaford, Del.) andmaintained under specific pathogen-free conditions. The mice were fedPicoLab® irradiated mouse chow (Lab Diet, Richmond, Ind.) and autoclavedwater was freely available. All procedures were carried out under theinstitutional guidelines of TGen Drug Development Services InstitutionalAnimal Care and Use Committee (Protocol #06001, Approved January 2006).

A2780 Human Ovarian Tumor Xenograft

Ninety mice were inoculated subcutaneously in the right flank with 0.1ml of a 50% RPMI/50% Matrigel™ (BD Biosciences, Bedford, Mass.) mixturecontaining a suspension of A2780 tumor cells (approximately 1×10⁷cells/mouse). Four days following inoculation, tumors were measuredusing vernier calipers and tumor weight was calculated using the animalstudy management software, Study Director V1.6.80 (Study Log) (CancerRes 59: 1049-1053). Seventy mice with an average group tumor size of 108mg, with mice ranging from 61 to 184 mg, were pair-matched into sevengroups of ten by random equilibration using Study Director (Day 1). Bodyweights were recorded when the mice were pair-matched and then takentwice weekly thereafter in conjunction with tumor measurementsthroughout the study. Gross observations were made at least once a day.The dosing schedule, indicated on Table 39, shows that with minorvariation COTI-2 was dosed every other day 3 times weekly and Doxil® (2mg/kg) was administered intravenously on Day 1 at a 10-ml/kg dosevolume. The mice were sacrificed by regulated CO₂ when the mean tumorvolume of the control group reached approximately 2000 mg.

Data and Statistical Analysis

Mean tumor growth inhibition (TGI) was calculated utilizing thefollowing formula:

${TGI} = {\left\lbrack {1 - \frac{\left( {{\overset{\_}{\chi}}_{{Treated}_{({Final})}} - {\overset{\_}{\chi}}_{{Treated}_{({{Day}\; 1})}}} \right)}{\left( {{\overset{\_}{\chi}}_{{Control}_{({Final})}} - {\overset{\_}{\chi}}_{{Control}_{({{Day}\; 1})}}} \right)}} \right\rbrack \times 100\%}$

Tumors that regressed from the Day 1 starting size were removed from thecalculations. All statistical analyses in the xenograft study wereperformed with GraphPad Prism® v4 software. Differences in Day 17 tumorweights were confirmed using Analysis of Variance (ANOVA) with theTukey's Post Test. In addition to an ANOVA, a One-tailed Student's Ttest was used to compare the vehicle only group to each of thecombination agent groups and single agent Doxil®. Increase in survivalfraction was confirmed by the log rank test.

Results and Discussion:

This study determined the single agent efficacy of COTI-2 in the A2780human ovarian tumor xenograft model. COTI-2 was also studied incomparison, as well as in combination, with the standard agent Doxil®.Efficacy was assessed by comparison of mean tumor weights.

Overall, each test agent was moderately tolerated with a weight loss ofup to ≈10% (Table 39). Single agent COTI-2 and Doxil® treatments didresult in tumor growth inhibition (up to 25.76%). Furthermore, thecombination agent treatment groups exhibited better tumor growthinhibition (up to 53.80%) relative to the single agent treatment groups.

TABLE 39 Summary of the study parameters and results. Day 17 Mean DayMaximum tumor 17 Day Dose Schedule weight loss weight TGI 17 Group N(mg/kg) Route (Day) (%) (Day #) (mg) (%) CS* Vehicle 10 — IV 1, 3, 5, 7,8, 10.84% (Day 15) 2,150.7 ± 352.5 — 0 Control 10, 12, 15, 16, 17 COTI-210 12.5 IV 1, 3, 5, 7, 8, 10.35% (Day 11) 1,699.2 ± 241.4 22.04 0 10,12, 15, 16, 17 COTI-2 10 25.0 IV 1, 3, 5, 7, 8,  9.37% (Day 8) 1,738.4 ±216.0 20.12 0 10, 12, 15, 16, 17 COTI-2 10 50.0 IV 1, 3, 5, 7, 8,  8.65%(Day 8) 1,622.8 ± 403.0 25.76 0 10, 12, 15, 17 Doxil ® 10 2.0 IV 1 3.76% (Day 4) 1,607.1 ± 343.4 25.35 0 COTI-2 10 12.5 IV 1, 2, 5, 8, 10, 7.67% (Day 11) 1,045.3 ± 305.7 47.44 1/10 Doxil ® 2.0 IV 12, 15, 17 1COTI-2 10 25.0 IV 1, 2, 5, 8, 10,  7.68% (Day 8) 1,046.0 ± 261.3 53.80 0Doxil ® 2.0 IV 12, 15, 17 1 *CS = Complete Tumor Shrinkage

The combination of COTI-2 and Doxil® resulted in positive interactiontrends (FIG. 54). In fact, the combination agents exhibitedsignificantly smaller tumors compared to the vehicle control group(P<0.05), whereas there was no statistically significant difference intumor size of the Doxil® single agent group relative to the vehiclecontrol group (P>0.05), except at day 4. These data indicate that theCOTI-2 and Doxil® combination agents perform better than the Doxil®single agent in this xenograft model.

Example 27 Evaluation of COTI-2 as a Single Agent and in Combinationwith Erbitux® in the HT-29 Human Colon Tumor Xenograft Model

The purpose of this study was to evaluate the activity of COTI-2 as asingle agent and in combination with Erbitux® in the HT-29 human colontumor xenograft model.

Materials and Methods:

Reagents and Compounds:

COTI-2 mono-HCl salt (Lot#CKL-2-148) stored at −20° C. until ready foruse. A stock solution of COTI-2 was made at a concentration of 12 mg/ml;235 mg of COTI-2 was mixed with 4.84 g of Captisol® (Lot#CY-04A-05006Cyclex Pharmaceuticals; Lenexa, Kans.) in 10.0 ml of Sterile Water forInjection (Hospira, Inc., Lake Forest, Ill.). The solution was preparedin a 60 ml amber vial and left to stir at 400 rpm for 1 h. The solutionwas then filtered with a 0.2 μm PVDF filter to achieve the 12 mg/mlstock concentration. The stock was further diluted to 6 mg/ml with 50 mMCaptisol® solution in sterile water (at pH 3.0). The 6 mg/ml solutionwas also filtered with a 0.2 μm PVDF filter and was administeredintravenously with respect to the individual groups. Erbitux®(Lot#07C00373B), was manufactured by Bristol-Myers Squibb Company(Princeton, N.J.), and was stored at 4° C. until used. Erbitux® wasgiven intraperitoneally at a volume of 0.5 ml per mouse to deliver 1mg/dose every three days for five treatments (q3d×5). All preparationswere made fresh prior to their administration.

Cell Culture

The HT-29 human colon tumor cell line was received from American TypeCulture Collection (ATCC, Manassas, Va.). Cultures were maintained inRPMI 1640 (Hylcone Labs, Logan, Utah) supplemented with 5% fetal bovineserum, and housed in a 5% CO₂ atmosphere. The cultures were expanded intissue culture flasks at a 1:4 split ratio until a sufficient amount ofcells were harvested.

Animals

Female athymic nude mice (CrTac: NCR-Foxn1^(nu)) were supplied byTaconic (Germantown, N.Y.). Mice were received at four weeks of age. Allmice were acclimated for seven days prior to handling. The mice werehoused in microisolator cages (Lab Products, Seaford, Del.) andmaintained under specific pathogen-free conditions. The mice were fedPicoLab® irradiated mouse chow (Lab Diet, Richmond, Ind.) and autoclavedwater was freely available. All procedures were carried out under theinstitutional guidelines of TGen Drug Development Services InstitutionalAnimal Care and Use Committee (Protocol #09002, Approved February 2009).

HT-29 Human Colon Tumor Xenograft

Eighty five mice were inoculated subcutaneously in the right flank with0.1 ml of a 50% RPMI/50% Matrigel™ (BD Biosciences, Bedford, Mass.)mixture containing a suspension of HT-29 tumor cells (approximately5×10⁶ cells/mouse). Seven days following inoculation, tumors weremeasured using vernier calipers and tumor weight was calculated usingthe animal study management software, Study Director V.1.6.80 (StudyLog) (Cancer Res 59: 1049-1053). Seventy mice with average group tumorsizes of 189 mg, with mice ranging from 123 to 252 mg, were pair-matchedinto seven groups of ten by random equilibration using Study Director(Day 1). Body weights were recorded when the mice were pair-matched andthen taken twice weekly thereafter in conjunction with tumormeasurements throughout the study. Gross observations were made at leastonce a day. On Day 1 all groups were dosed intravenously and/orintraperitoneally with respect to their group (See Table 40). Thevehicle, COTI-2 (12.5 mg/kg) and COTI-2 (25 mg/kg) groups were doseddaily 5 times per week via IV injection, except for the first week.COTI-2 in the remaining groups was dosed 3 times per week on every otherday via IV injection. Erbitux® (1 mg/dose) was administeredintraperitoneally every three days for five treatments (q3d×5) at 0.5ml/mouse dose volume. The mice were sacrificed by regulated CO₂ when theindividual mouse tumor volume reached approximately 2000 mg.

Data and Statistical Analysis

Mean tumor growth inhibition (TGI) was calculated utilizing thefollowing formula:

${TGI} = {\left\lbrack {1 - \frac{\left( {{\overset{\_}{\chi}}_{{Treated}_{({Final})}} - {\overset{\_}{\chi}}_{{Treated}_{({{Day}\; 1})}}} \right)}{\left( {{\overset{\_}{\chi}}_{{Control}_{({Final})}} - {\overset{\_}{\chi}}_{{Control}_{({{Day}\; 1})}}} \right.}} \right\rbrack \times 100\%}$

Tumors that regressed from the Day 1 starting size were removed from thecalculations. All statistical analyses in the xenograft study wereperformed with GraphPad Prism® v4 software. Differences in Day 15 tumorweights were confirmed using the Analysis of Variance (ANOVA) with theTukey's Post Test. In addition to an ANOVA, a One-tailed Student's Ttest was used to compare the vehicle only group to each of thecombination agent groups and single agent Erbitux® group. Increase insurvival fraction was confirmed by the log rank test.

Results and Discussion:

This study determined the single agent efficacy of COTI-2 in the HT-29human colon tumor xenograft model. COTI-2 was also studied incombination with the standard agent, Erbitux®.

Erbitux® was well-tolerated and produced no weight loss alone.Interestingly, weight loss was also not observed in the COTI-2 (12.5mg/kg) and Erbitux® combination group in which COTI-2 was administered 3times per week on every other day.

The combination agent groups exhibited a significant tumor reductionearly during the study (days 4 & 8) relative to the vehicle controlgroup (FIG. 55). However, there was no significant difference in tumorweight between the single agent Erbitux® group and the vehicle controlgroup. These data suggest that the COTI-2 and Erbitux® combinationperforms better than the single agent COTI-2 or Erbitux®.

Example 28 Evaluation of COTI-2 as a Single Agent and in Combinationwith Erbitux® in the HCT-116 Human Colon Tumor Xenograft Model

The purpose of this study was to evaluate the activity of COTI-2 as asingle agent and in combination with Erbitux® in the HCT-116 human colontumor xenograft model.

Materials and Methods:

Reagents and Compounds

COTI-2 mono HCl salt (Lot#CKL-2-148) was stored at −20° C. until readyfor use. A stock solution of COTI-2 was made at a concentration of 12mg/ml; 235 mg of COTI-2 was mixed with 4.84 g of Captisol®(Lot#CY-04A-05006 Cydex Pharmaceuticals; Lenexa, Kans.) in 10.0 ml ofSterile Water for Injection (Hospira, Inc., Lake Forest, Ill.). Thesolution was prepared in a 60 ml amber vial and left to stir at 400 rpmfor 1 h. The solution was then filtered with a 0.2 μm PVDF filter toachieve the 12 mg/ml stock concentration. The stock was further dilutedto 6 mg/ml with 50 mM Captisol® solution in sterile water (at pH 3.0).The 6 mg/ml solution was also filtered with a 0.2 μm PVDF filter and wasgiven intravenously with respect to the individual groups. Erbitux®(Lot#07C00373B), was manufactured by Bristol Meyers Squibb Company(Princeton, N.J.), and was stored at 4° C. until used. Erbitux® wasgiven intraperitoneally at a volume of 0.5 ml per mouse to deliver a 1mg/dose every three days for four treatments (q3d×4). All preparationswere made fresh prior to their administration.

Cell Culture

The HCT-116 human colon tumor cell line was received from American TypeCulture Collection (ATCC, Manassas, Va.). Cultures were maintained inRPMI 1640 (Hylcone Labs, Logan, Utah) supplemented with 5% fetal bovineserum, and housed in a 5% CO₂ atmosphere. The cultures were expanded intissue culture flasks at a 1:6 split ratio until a sufficient amount ofcells were harvested.

Animals

Female athymic nude mice (CrTac: NCR-Foxn1^(nu)) were supplied byTaconic (Germantown, N.Y.). Mice were received at four weeks of age. Allmice were acclimated for seven days prior to handling. The mice werehoused in microisolator cages (Lab Products, Seaford, Del.) andmaintained under specific pathogen-free conditions. The mice were fedPicoLab® irradiated mouse chow (Lab Diet, Richmond, Ind.) and autoclavedwater was freely available. All procedures were carried out under theinstitutional guidelines of TGen Drug Development Services InstitutionalAnimal Care and Use Committee (Protocol #06001, Approved January 2006).

HCT-116 Human Colon Tumor Xenograft

Ninety mice were inoculated subcutaneously in the right flank with 0.1ml of a 50% RPMI/50% Matrigel™ (BD Biosciences, Bedford, Mass.) mixturecontaining a suspension of HCT-116 tumor cells (approximately 5×10⁶cells/mouse). Three days following inoculation, tumors were measuredusing vernier calipers and tumor weight was calculated using the animalstudy management software, Study Director V.1.6.80 (Study Log) (CancerRes 59: 1049-1053). Seventy mice with average group tumor sizes of 136mg, with mice ranging from 73 to 194 mg, were pair-matched into sevengroups of ten by random equilibration using Study Director (Day 1). Bodyweights were recorded when the mice were pair-matched and then takentwice weekly thereafter in conjunction with tumor measurementsthroughout the study. Gross observations were made at least once a day.On Day 1 all groups were dosed intravenously and/or intraperitoneallywith respect to their assigned group (See Table 40). The COTI-2 singleagent groups were treated 3 times per week on every other day for thefirst week of the study then dosed 5 times per week for the remainder ofthe study. In the COTI-2 and Erbitux® combination treatment groups,COTI-2 was administered 3 times per week on every other day. Erbitux® (1mg/dose) was administered intraperitoneally every three days for fivetreatments (q3d×5) at 0.5 ml/mouse dose volume. The mice were sacrificedby regulated CO₂ when the individual mouse tumor volume reachedapproximately 2000 mg.

Data and Statistical Analysis

All statistical analyses in the xenograft study were performed withGraphPad Prism® v4 software. Increase in survival fraction was confirmedby the log rank test. A Student's T test was used to evaluate differencein tumor weight among the treatment groups.

Results and Discussion:

This study determined the single agent efficacy of COTI-2 in the HCT-116human colon tumor xenograft model. COTI-2 was also studied incombination with the standard agent, Erbitux®.

The HCT-116 model is an aggressive model in terms of growth kinetics.Therefore, it is likely that treatment was less tolerated due to theearly onset of cachexia observed in colon tumor xenograft models. Therewas an increase in body weight loss by the test agent groups compared tovehicle control (Table 40); in addition, it may be possible that theCOTI-2 treated mice experienced a drug accumulation effect. Erbitux® waswell-tolerated and produced a moderate weight loss, which may have beena result of tumor burden.

TABLE 40 Summary of the study parameters and results. Day 15 MaximumMean Mean weight tumor survival Dose Schedule loss (%) weight (Day ±Group N (mg/kg) Route (Day) (Day #) (mg) SEM) Deaths Vehicle 10 — IV 1,3, 4, 5, 8, 10,  17.1% 1,426.8 ± 141.5 11.5 ± 1.03 3 Control 11, 12, 15(Day 15) COTI-2 10 12.5 IV 1, 3, 4, 5, 8, 10, 20.96% 1,471.3 ± 155.912.8 ± 0.76 1 11, 12, 15, 16, 17 (Day 17) COTI-2 10 25.0 IV 1, 3, 4, 5,8, 10, 11.12% 1,376.9 ± 139.4 11.5 ± 0.65 2 11, 12, 15 (Day 8) COTI-2 1050.0 IV 1, 3, 5, 8, 10, 11, 18.43% 1,353.4 ± 127.3 12.6 ± 0.83 2 12, 15,16, 17, (Day 22) 18, 19, 22 Erbitux ® 10 2.0 IP 1, 4, 7, 10, 13 17.15%1,196.2 ± 104.1 13.6 ± 0.73 0 (Day 15) COTI-2 10 12.5 IV 1, 3, 5, 8, 10,12, 15.87% 1,126.2 ± 147.7 13.5 ± 1.19 1 Erbitux ® 2.0 IP 15, 17, 19, 22(Day 15) 1, 4, 7, 10, 13 COTI-2 10 25.0 IV 1, 3, 5, 8, 10, 12, 19.85%1,221.2 ± 93.7  14.2 ± 0.74 0 Erbitux ® 2.0 IP 15, 17 (Day 17) 1, 4, 7,10, 13

FIG. 56 shows a trend with respect to a decrease in tumor size with theCOTI-2 (12.5 mg/kg) and Erbitux® (1 mg/dose) combination regimen. TheCOTI-2 (25 mg/kg) and Erbitux® (1 mg/dose) combination regimen produceda survival fraction that was significantly increased from the vehiclecontrol group (p<0.05). There was no significant difference in the meansurvival of the Erbitux® only treated group when compared to the vehiclecontrol group. These data indicate that the COTI-2 and Erbitux®combination increases survival.

What is claimed is:
 1. A therapeutically effective composition for usein the treatment of cancer comprising an anti-cancer agent and atherapeutically effective amount of a compound comprising a compound ofFormula I:

and/or a pharmaceutically-acceptable salt thereof; wherein: R₁ and R₂together form a substituted or unsubstituted polycyclic ring comprisingat least two ring systems, said at least two ring systems comprising afirst ring system bonded to C1 and a second ring system fused to thefirst ring system, wherein: the first ring system is a substituted orunsubstituted aromatic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstitutedheteroaromatic group, a substituted or unsubstituted carbocyclic group,or a substituted or unsubstituted heterocyclic group; or the first ringsystem is a substituted or unsubstituted heteroaromatic group, thesecond ring system is a substituted or unsubstituted aromatic group, asubstituted or unsubstituted) heteroaromatic group, a substituted orunsubstituted carbocyclic group, or a substituted or unsubstitutedheterocyclic group; or the first ring system is a substituted orunsubstituted saturated carbocyclic group, the second ring system is asubstituted or unsubstituted aromatic group, a substituted orunsubstituted unsaturated carbocyclic group, a substituted orunsubstituted heterocyclic group, or a substituted or unsubstituted ringB:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; or the first ring system is a substituted or unsubstitutedunsaturated carbocyclic group, the second ring system is a substitutedor unsubstituted aromatic group, a substituted or unsubstitutedcarbocyclic group, a substituted or unsubstituted heterocyclic group, ora substituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; or the first ring system is a substituted or unsubstitutedheterocyclic group, the second ring system is a substituted orunsubstituted heteroaromatic group, a substituted or unsubstitutedcarbocyclic group, or a substituted or unsubstituted heterocyclic group;and R₃ to R₁₁ are each independently selected from H, a substituted orunsubstituted hydrocarbon group, a substituted or unsubstitutedheterogeneous group, a substituted or unsubstituted carbocyclic group, asubstituted or unsubstituted heterocyclic group, substituted orunsubstituted aromatic, or a substituted or unsubstitutedheteroaromatic; R₁₂ is selected from H or a hydrocarbyl group; Y isselected from a heteroatom or a carbon atom; A is selected from asubstituted or unsubstituted hydrocarbon group, a substituted orunsubstituted heterogeneous group, a substituted or unsubstitutedcarbocyclic group, a substituted or unsubstituted heterocyclic group,substituted or unsubstituted aromatic, or a substituted or unsubstitutedheteroaromatic; and n is 0 or 1; wherein the composition produces asynergistic therapeutic effect as compared to sole administration ofeither the anti-cancer agent or the compound.
 2. The compositionaccording to claim 1, wherein the compound is an mTOR-Rictor complexinhibitor.
 3. The composition according to claim 1, wherein theanti-cancer agent is an mTOR-Raptor complex inhibitor.
 4. Thecomposition according to claim 1, wherein the anticancer agent is acytotoxic agent.
 5. The composition according to claim 4, wherein thesynergistic effect is reduction or prevention of resistance to thecytotoxic agent.
 6. The composition according to claim 1, wherein theanti-cancer agent is selected from the group consisting of cisplatin,rapamycin, tecrolimus, temsirolimus, paclitaxel, erlotinib, cetuximabdoxorubicin, and combinations thereof.
 7. The composition according toclaim 1, wherein the cancer is treatable by inhibition of mTOR.
 8. Thecomposition according to claim 1, wherein the amount of the anti-canceragent is selected to lower overall toxicity as compared toadministration of the anti-cancer agent alone in an amount sufficient toachieve substantially the same treatment effect on cancerous cells. 9.The composition according to claim 1, wherein the dose of at least oneof the anti-cancer agent or the compound is selected to increase theoverall treatment effect on cancerous cells as compared toadministration of the anti-cancer agent alone in an amount producingsubstantially the same toxicity.
 10. The composition according to claim1, wherein the first ring system is a substituted or unsubstitutedheterocyclic group, the second ring system is a substituted orunsubstituted heteroaromatic group, a substituted or unsubstitutedcarbocyclic group, or a substituted or unsubstituted heterocyclic group.11. The composition according to claim 10, wherein n is
 0. 12. Thecomposition according to claim 10, wherein n is 1 and A is a substitutedor unsubstituted heteroaromatic group.
 13. The composition according toclaim 12, wherein A is a pyridinyl group.
 14. The composition accordingto claim 10, wherein Y is a nitrogen atom.
 15. The composition accordingto claim 14, wherein R₇ is a substituted or unsubstituted alkyl group ora substituted or unsubstituted heteroaromatic group and R₃ to R₆ and R₈to R₁₂ are each independently selected from H or a substituted orunsubstituted hydrocarbon group.
 16. The composition according to claim15, wherein R₇ is the substituted or unsubstituted alkyl group or asubstituted or unsubstituted pyridyl group and R₃ to R₆ and R₈ to R₁₂are each H.
 17. The composition of claim 1, wherein the compound isselected from:

and/or a pharmaceutically-acceptable salt thereof.
 18. The compositionaccording to claim 17, wherein the compound of Formula I is:

and/or a pharmaceutically-acceptable salt thereof.
 19. The compositionaccording to claim 17, wherein the compound of Formula I is:

and/or a pharmaceutically-acceptable salt thereof.
 20. Apharmaceutically-acceptable oxalate or tartrate salt of a compound ofFormula I:

and/or optical isomer thereof; wherein: R₁ and R₂ together form asubstituted or unsubstituted polycyclic ring comprising at least tworing systems, said at least two ring systems comprising a first ringsystem bonded to C1 and a second ring system fused to the first ringsystem, wherein: the first ring system is a substituted or unsubstitutedaromatic group, the second ring system is a substituted or unsubstitutedaromatic group, a substituted or unsubstituted heteroaromatic group, asubstituted or unsubstituted carbocyclic group, or a substituted orunsubstituted heterocyclic group; or the first ring system is asubstituted or unsubstituted heteroaromatic group, the second ringsystem is a substituted or unsubstituted aromatic group, a substitutedor unsubstituted heteroaromatic group, a substituted or unsubstitutedcarbocyclic group, or a substituted or unsubstituted heterocyclic group;or the first ring system is a substituted or unsubstituted saturatedcarbocyclic group, the second ring system is a substituted orunsubstituted aromatic group, a substituted or unsubstituted unsaturatedcarbocyclic group, a substituted or unsubstituted heterocyclic group, ora substituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; or the first ring system is a substituted or unsubstitutedunsaturated carbocyclic group, the second ring system is a substitutedor unsubstituted aromatic group, a substituted or unsubstitutedcarbocyclic group, a substituted or unsubstituted heterocyclic group, ora substituted or unsubstituted ring B:

wherein X₁ to X₆ are each independently selected from carbon or aheteroatom; or the first ring system is a substituted or unsubstitutedheterocyclic group, the second ring system is a substituted orunsubstituted heteroaromatic group, a substituted or unsubstitutedcarbocyclic group, or a substituted or unsubstituted heterocyclic group;and R₃ to R₁₁ are each independently selected from H, a substituted orunsubstituted hydrocarbon group, a substituted or unsubstitutedheterogeneous group, a substituted or unsubstituted carbocyclic group, asubstituted or unsubstituted heterocyclic group, substituted orunsubstituted aromatic, or a substituted or unsubstitutedheteroaromatic; R₁₂ is selected from H or a hydrocarbyl group; Y isselected from a heteroatom or a carbon atom; A is selected from asubstituted or unsubstituted hydrocarbon group, a substituted orunsubstituted heterogeneous group, a substituted or unsubstitutedcarbocyclic group, a substituted or unsubstituted heterocyclic group,substituted or unsubstituted aromatic, or a substituted or unsubstitutedheteroaromatic; and n is 0 or
 1. 21. The composition according to claim1, wherein the anti-cancer agent is more than one anti-cancer agent. 22.The composition according to claim 1 further comprising gemcitabine. 23.The composition according to claim 6 further comprising gemcitabine.